www.sciencemag.org/content/351/6280/aad6253/suppl/DC1

Supplementary Materials for

Design and synthesis of a minimal bacterial genome Clyde A. Hutchison III,*† Ray-Yuan Chuang,† Vladimir N. Noskov, Nacyra Assad-

Garcia, Thomas J. Deerinck, Mark H. Ellisman, John Gill, Krishna Kannan, Bogumil J. Karas, Li Ma, James F. Pelletier, Zhi-Qing Qi, R. Alexander Richter, Elizabeth A. Strychalski, Lijie Sun, Yo Suzuki, Billyana Tsvetanova, Kim S. Wise, Hamilton O.

Smith, John I. Glass, Chuck Merryman, Daniel G. Gibson, J. Craig Venter,*

*Corresponding author. E-mail: chutchis@jcvi.org (C.A.H.); jcventer@jcvi.org (J.C.V.) †These authors contributed equally to this work.

Published 25 March 2016, Science 351, aad6253 (2016)

DOI: 10.1126/science.aad6253

This PDF file includes:

Materials and Methods Figs. S1 to S21 Tables S1 to S15 Full Reference List

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/content/351/6280/aad6253/suppl/DC1)

Database S1 as an Excel file

   

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Materials and Methods Procedure for identification of non-essential genes by Global Tn5 transposon mutagenesis. A sequence map of the Tn5 puromycin resistance (Tn5 puro) transposon is shown in Fig S1. To make active transposomes, Tn5 transposase was reacted with Tn5 puro DNA according to Epicentre instructions. Briefly, 2 µl of Tn5 transposon DNA (0.1 µg/µl), 2 µl of glycerol, and 4 µl of Tn5 transposase were mixed together by vortexing, incubated for 30 min at room temperature, and then stored at -20 °C.

M. Mycoides cells were grown in SP4 media to pH 6.3-6.8. A 40ml culture was centrifuged at 10 °C for 15 min at 4700 rpm in a 50 ml tube. The pellet was resuspended in 3 ml of Tris 10 mM, sucrose 0.5 M, pH 6.5 (Buffer S/T). The resuspended cells were centrifuged for 15 min at 4700 rpm at 10 ºC. The pellet, still in the 50ml tube, was suspended in 250 µl of 0.1 M CaCl2 and incubated for 30 min on ice. Transposomes (2µl) and yeast tRNA (Life Technologies) 10mg/ml (1µl) were added and mixed gently with the cells. Two mls of 70% Polyethylene glycol (PEG) 6000 (Sigma) dissolved in S/T buffer was added. The suspension was incubated at room temperature with gentle mixing, and then 20 ml S/T buffer was added and mixed well. Cells were centrifuged at 8 °C for 15 min at 10,000 x g. The supernatant was discarded and the tube drained well to remove the PEG. The cells were resuspended in 1 ml of warm SP4 media, incubated for 2 hours at 37 °C, and then plated on SP4 agar containing 10 µg/ml of Puromycin (Puro) (Sigma). Colonies were generally visible after 3 to 4 days at 37 °C. The various steps in the Tn5 mutagenesis procedure are illustrated in Fig S2.

Colonies (> 40,000 total) from several plates were suspended in 22ml of SP4 media containing puromycin 10µg/ml (P0). Fifty µl of P0 cells were diluted into 50 ml SP4 puro media and incubated at 37 °C for 24 hours (P1 passage). P1 cells (3µl) were diluted into 50 ml of fresh SP4 puro medium and grown for 24h (P2). Two more serial passages were done to yield P3 and P4 cell cultures (Fig S2).

P4 cells were centrifuged at 4700 rpm for 15 min. The cell pellet was resuspended in 300 µl of 0.1 M NaCl, 20 mM NaEDTA solution (pH 8.0) in a 1.5 ml of Eppendorf tube. SDS 10% (30 µl) and proteinase K 5 µg (USB Corporation) were added and mixed well. The tube was incubated overnight at 37 °C. The mixture was extracted with 330 µl of aqueous phenol (Sigma) and then centrifuged at 15,000 RPM for 5 min. The supernatant was transferred to a new 1.5 ml tube and extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) (Sigma). After centrifugation the supernatant was transferred to a new 1.5 ml tube. A 1/10 volume of 3 M sodium acetate (Sigma) was added and the DNA was ethanol precipitated. After centrifugation at 8,000 Xg for 5 min the pellet was dissolved in 200 µl of TE buffer (pH 8.0) (Teknova). One µl of RiboShredder™ RNase Blend (Epicentre) was added and the mixture incubated it at 37 °C for 1 hour. The DNA was then extracted with phenol/chloroform, ethanol precipitated, and the pellet dissolved in 200 µl of TE. P0 DNA was similarly prepared from a sample of P0 cells (Fig S2).

DNA enriched for transposon/genomic DNA junctions was obtained by inverse PCR ((5), Fig S2) as follows. P0 or P4 DNA (40 µl) in 0.5 ml of TE was sheared in a

   

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Nebulizer (Invitrogen™) at 15 lb/in2 for 30sec to obtain 1.5 to 2 kb fragments of DNA. The DNA was ethanol-precipitated, dissolved in 100 µl TE, diluted with 100 µl of 2X BAL31 nuclease buffer and digested with 1 µl of BAL31 nuclease (NEB, 1U/µl) for 5 min at room temperature to produce blunt-ended DNA. After phenol extraction and ethanol precipitation, DNA was circularized using quick DNA ligase (NEB). Ligase was inactivated at 65°C for 20 min. The library was amplified by PCR using 2.5 µl of SqFP and SqRP primers (10 µM each), 20 µl of ligation reaction, and 25 µl of Phusion® High-Fidelity PCR Master Mix (NEB). The cycling conditions were one cycle at 98°C for 30 sec, 29 cycles at 98 °C for 15sec, 58 °C for 20 sec, 72 °C for 3 min with a final extension for 5 min at 72 °C. The PCR product, ranging from ≈ 0.5 to 1 kb, was purified using phenol/chloroform extraction and ethanol precipitation and dissolved in TE at a concentration of ~40 ng/µl.

Miseq sequencing Paired-end libraries for Miseq sequencing were constructed from template DNA according to the manufacturer’s protocol (Nextera XT DNA, Illumina, San Diego, CA, USA). Briefly this method involved using a transposase loaded with adapter oligos to simultaneously fragment the input DNA and ligate adapter sequences in a single reaction. The adapter sequences were then used to amplify the DNA in a reduced-cycle PCR reaction. During the PCR reaction, unique index sequences were added to both ends of the DNA to allow for dual-indexed sequencing of the pooled libraries. PCR cleanup was performed using a .5:1 ratio of Ampure XP (Beckman Coulter) to PCR reaction. Libraries were normalized following Illumina’s instructions for XT bead-based normalization. In preparation for cluster generation and sequencing, equal volumes of each normalized library was combined, diluted 25-fold in hybridization buffer, and heat denatured. The final library pool was sequenced according to standard protocols (MiSeq, Illumina, San Diego, CA, USA). Tn5 sequencing data analysis and gene classification. The sequence reads were searched for the 19-bp terminus of the Tn5 transposon followed by a 30-bp exact match to genome DNA sequence. In earlier mapping procedures we used BLAST to locate the insertion sites, but this led to a low background of erroneous site locations. These spurious insertions disappeared when we shifted to the requirement for an exact 30-bp match immediately following the 19-bp Tn5 terminus. The junction point between the Tn5 sequence and the genome sequence was taken as the insertion point. A large number of insertions were found and there were a number of hot spots for insertion, but only the set of unique insertion coordinates were used. The scarless TREC deletion method for producing the D-series deletions. The scarless TREC (tandem repeat coupled with endonuclease cleavage) deletion method (37, 38) was used to generate a series of reduced genomes in yeast. Six insertion elements (IS) and two genes (MMSYN1_0460 and _0463) flanking one of the IS elements were sequentially deleted in the genome of JCVI-syn1.0 ΔRE (39) to produce the Syn1.0∆RE∆IS genome. Based on Tn5 insertion data, twenty-two putatively non-essential gene clusters were selected and subjected to deletion sequentially in syn1.0∆RE∆IS to produce 22 strains (D1 to D22). In each round of deletion, the genome

   

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was tested for viability by transplantation. If the growth rate approximately equaled that of syn1.0∆RE∆IS, then the next round was begun. Detailed information regarding each of the sequential deletion strains is shown in Table S6. Scarless deletions were produced using the TREC method (Fig S8). The design of a knock-out cassette is described previously except the length of a repeated sequence was reduced to 50 bp. Unique knock-out cassettes were produced by 2 rounds of PCR using the Advantage HD Polymerase (Clontech) according to the manufacturer’s instructions. The first round of PCR was performed for 18 cycles using the pCORE3 plasmid as a DNA template and primer pair 1 (Table S11). The second round of PCR was performed for 22 cycles using the first round PCR product as DNA template and primer pair 2 (Table S11). Chimeric primers in the first round PCR generate a CORE cassette (38) flanked by a 50 bp repeated sequence on the 5’ end and 50 bp sequences for homologous recombination on the 3’ end. Chimeric primers in the second round PCR generate a final knock-out cassette containing, on the 5’ to 3’ end, 50 bp for homologous recombination, 50 bp repeated sequence, and 50 bp for homologous recombination (Fig S8). The second round of PCR product was purified by the MinElute PCR Purification Kit (Qiagen). Approximately 0.5 to 1 µg purified PCR product was used for yeast transformation by the lithium acetate method. The procedure of TREC deletion and cassette recycling was described previously (38). Briefly after transformation, cells were plated on synthetic defined minimal dropout medium, SC-URA. Clones were screened by PCR analysis for the boundaries between the cassette and target site. Positive clones were grown on YEPG media to induce the expression of endonuclease I-SceI. A double strand cleavage by the endonuclease promoted a homologous recombination between two repeated sequences, leading to removal of the CORE cassette. After induction, cells were grown in SC-HIS+ 5-FOA to select for the removal of the cassette. The precise recycling of the cassette was verified by junction PCR (Fig S8). Primers used for screening knock-out and CORE3 cassette recycling are listed in Table S11. Method for creating 8 genomic segments flanked by NotI sites to facilitate design and genome assembly. The syn1.0∆RE∆IS strain (Table S6) was used as the parental strain to create eight NotI strains (Fig S9). Eight genomes were modified by the TREC method to engineer two NotI recognition sites (GCGGCCGC) in yeast. The locations of NotI sites were either in an intergenic region or Tn5-defined non-essential gene coding region. NotI sites encompass approximately 1/8th of the genome (Fig S9A). A non-essential region (24,916-bp) was not included in the design of the 1st 1/8th genome (between NotI -8 and NotI -1), thus 200-bp overlapping the adjacent segment 1 was introduced to the end of segment 8 next to the NotI site (Fig S9A).

Each of the eight 1/8th genome segments (NotI -1 to NotI -8) overlaps adjacent segments by 200 base pair (bp) allowing assembly of the segments into a complete genome in yeast via homologous recombination (Fig S9B). These engineered genomes were transplanted into M. capricolum recipient cells to produce 8 NotI mycoides strains (NotI -1 to -8 strains). To assemble the mycoides genome, genome DNA was prepared in agarose plugs and digested with restriction enzyme NotI, which generated two fragments, the 1/8th and the 7/8th genome. The 1/8th genome was separated from 7/8th genome by

   

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field-inversion gel electrophoresis (FIGE), shown in Fig S9C. The 1/8th genome was then recovered by electro-elution from the agarose gel (Fig S9C). To assemble the mycoides genome, all 8 purified overlapping 1/8th genome segments were co-transformed into yeast spheroplasts and selected on appropriate media. A complete genome assembly was evaluated by multiplex PCR (MPCR) and sized by electrophoresis.

Initially, assembled genomes were selected inefficiently by using the yeast his3 marker on segment 1. To enhance the efficiency of obtaining complete genome assemblies it was desirable to insert an additional selectable marker elsewhere than in NotI -1. We knew that the large cluster of genes MMSYN1 0550 to 0591 in NotI -6 was dispensable so we replaced this cluster with the yeast selectable marker MET14. At the same time this substantially reduced the size of NotI -6 (Fig S9A). Double MET14, his3 selection substantially improved the odds of obtaining completely assembled genomes. Method for construction of single-segment-RGD genomes (1/8th RGD +7/8th syn1.0). To aid in testing the functionality of each RGD synthetic segment, semi-RGD genomes consisting of 1/8th RGD piece and 7/8th syn1.0 genome were constructed and transplanted. A swapping approach, based on recombinase-mediated cassette exchange (RMCE), was developed to improve the efficiency of genome construction (18). Eight landing pad yeast strains containing truncated genomes were constructed by replacing individual 1/8th genome segments with a landing pad cassette containing a truncated URA3 gene and MET14 marker flanked by two mutant loxP sites (Fig S10). These 8 deleted segments were the same regions of the 8 NotI segments described in the Eight- NotI -strains strategy. To build a semi-RGD genome, a landing pad strain was mated with a corresponding opposite mating type yeast strain containing a donor vector with a RGD segment flanked by another two mutant loxP sites. Diploid strains were selected, followed by galactose induction to trigger the exchange of the RGD segment with the landing pad via the cre recombinase (Fig S10). The structure of the semi-RGD genome was first verified by PCR analysis for the boundaries between the RGD segment and 7/8th syn1.0 genome. The PCR products were further digested with NotI to confirm that the 1/8th RGD segment is flanked with NotI sites as designed. In general, the successful rate of swapping varied from 10% to 50 %.

At least two independent yeast clones were chosen for transplantation. All 8 semi-RGD genomes were able to produce colonies on SP4 medium containing tetracycline and X-gal at 37°C. Generally, colonies could be seen in 3 to 4 days after genome transplantation. The one exception was the transplanted cell from the RGD1.0-6 + 7/8th syn1.0 genome, which required about 10 days to produce a similar colony size as the others. Several faster growing cells were later isolated from the RGD1-6 transplant (See Fig. S18-S21). All 1/8th RGD segments can be released by NotI cleavage and purified for genome assembly.

Combinatorial assembly of segments to produce intermediate reduced genome designs (RGDs). All individual 1/8th RGD1.0 segments, together with appropriate 7/8th syn1.0 genomes, generated viable transplants, but the assembled complete RGD1.0 genome did not yield a viable transplant. To analyze potential synthetic growth defects or lethality among the 1/8th RGD1.0 segments, various combinations of segments were constructed. These

   

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intermediate RGDs, consisting of different combinations of 1/8th RGD1.0 segments, were assembled in yeast and then tested by transplantation. A number of transplants were obtained (Table S7B). Structures of the intermediate RGDs were determined from the MPCR amplicon patterns. In the first combinatorial assembly, an intermediate reduced genome, RGD2678, which contains 4 RGD1.0 segments (2, 6, 7, and 8) and 4 syn1.0 segments (1, 3, 4, and 5) exhibited an acceptable growth rate. The genome of RGD2678 was sequenced and subjected to Tn5 mutagenesis to find non-essential genes that might have converted to essential genes in this genomic background. From a second assembly, another intermediate RGD, RGD24*678, also exhibited an acceptable growth phenotype. Original MPCR data suggested that this genome contained 5 RGD1.0 segments (2, 4, 6, 7, and 8), but genome sequence data showed that segment 4 is a “hybrid” segment where approximately the first 1/3rd of syn1.0 segment 4 was substituted by RGD1.0 segment 4 sequence. This might result from a recombination between the RGD1.0 segment 4 and syn1.0 segment 4 during genome assembly in yeast. Instead of using the Tn5 mutagenesis approach, this clone was subjected to deletion targeting 39 gene clusters and single genes which had been removed in the design of RGD1.0- segments 1, 3, 4 and 5 (Table S8). The combined data from Tn5 mutagenesis of RGD2678 and the 39 individual deletions showed that 26 “n” genes among these 4 syn1.0 segments (1, 3, 4 and 5) had become “i” or “e” genes. Thus, RGD2678 was re-designed by adding back these 26 genes to RGD1.0 segments 1, 3, 4, and 5 to produce RGD2.0 segments 1, 3, 4, and 5 (Table S2 and S9). The new RGD2.0 design (Table S1) thus consisted of 4 segments of RGD1.0 (2, 6, 7, and 8), and 4 segments of RGD2.0 (1, 3, 4, and 5). RGD2.0 was assembled and multiple clones of complete genome assemblies were isolated and tested by genome transplantation. No viable transplant was obtained. To analyze potential synthetic growth defect and lethality among the RGD segments, 4 genomes consisting of 7 RGD segments and one syn1.0 segment (either 1, 3, 4, or 5) were assembled and transplanted. In the third assembly (Table S7B), three kinds of combinational genomes were able to produce transplants, except the genome assembled from 7 RGD and the syn1.0 segment 3 (Table S7B). Among three transplant clones, clone 49 with 7 RGD plus syn1.0 segment 5 had the smallest sized genome and yet exhibited a good growth rate. In parallel, a similar fourth assembly of 7 RGD segments and 1 syn1.0 segment was performed, except RGD2.0 segment 3 was replaced with the RGD1.0 segment 3s, a revised RGD1.0 segment 3 supplemented with three genes (Table S7A). Transplantation showed that only one genome, clone 48, containing 7 RGD and the syn1.0 segment 5 produced a transplant. The size of the clone 48 genome is about 10 kb smaller than that of the clone 49 genome since RGD2.0 segment 3 contains 7 more genes than does RGD1.0 segment 3s. Detailed derivation of the JCVI-syn2.0 genome from 7RGDs + syn1.0 segment 5. Among all mycoides strains with various intermediate RGD constructs, clone 48 had the smallest genome and an acceptable growth rate (≤120 min). Step-wise cluster deletions were used to explore a maximum reduction within syn1.0 segment 5 in clone 48. Of 39 genes and gene clusters (Table S10), 3 clusters (33, 36, and 37) located in syn1.0 segment 5 (Table S10) were selected for deletion by marker replacement. A number of deletion constructs were generated and transplanted. These constructs included all single cluster deletions, a double cluster deletion (∆33∆36), and a triple cluster deletion (∆33∆36∆37). All constructs were able to produce viable transplants. In addition, the genome with a

   

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deletion region, called cluster 0483-0492, covering the cluster 36, 37, and two genes (MMSYN1_0487 and 0488) between these 2 clusters was also able to produce viable transplants. At this point, after the triple cluster deletion, the size of the genome had been reduced to ~564 kb. The growth rate of the triple cluster deletion is about 120 min compared to 60 min for syn1.0. Syn1.0 contains two copies of the leucyl aminopeptidase gene, 0154 and 0190. These two genes were originally categorized as “n” genes, thus were deleted in the RGD1.0 design. However, data from Tn5 mutagenesis of the D10 genome (Table 6S) indicated that lack of both genes impaired cell growth. Replacement of genes 0487 and 0488 in clone 48 or its reduced derivatives with leucyl aminopeptidase gene 0154 resulted in an improved growth rate. Clone 48 with the above described deletions in the syn1.0 segment 5, including the gene 0154 add back resulted in the new reduced strain JCVI-syn2.0. Synthesis and assembly of reduced genomes A. Oligonucleotide Design Software. The oligonucleotide design software searches for a combination of parameters. These include dsDNA fragment overlap length, number of assembly stages, maximum fragment size, maximum number of fragments to be assembled per assembly stage and appended vector and restriction site sequences that will yield overlapping oligos that do not exceed 80 bases in length. We used an oligo overlap size equal to half the oligo length and dsDNA fragment overlap size of 40-80bp with the exception of the eighth molecules which contain defined 200-bp overlaps. At each stage of assembly, a unique set of vector sequences (30 bp) and restriction sites (8 bp) are appended to the 5’ and 3’ ends of each resulting fragment while ensuring the restriction sites are unique to each sequence. These appended sequences facilitated vector assembly for cloning or PCR amplification and insert release (i.e. removal of vector sequences) to expose overlaps for subsequent assembly stages (40). The oligonucleotide software was initially implemented as two WordPerfect macros: 1) Overlapping fragments.wcm, and 2) Oligonucleotides.wcm. The code for these macros is presented below. A streamlined version of the software is embedded in the Archetype® software package, which is commercially available through Synthetic Genomics, Inc. The Overlapping Fragments Macro was used to generate overlapping 7-kb cassettes starting from eighth molecule sequences. Vector and restriction site sequences were appended to the 7-kb cassettes and rerun through this same macro to generate ~1.4 kb dsDNA fragments. Vector and restriction site sequences were then appended to these sequences.  Application  (WordPerfect;  "WordPerfect";  Default!;  "EN")  Call(Lab1)  Quit()    Label(Lab1)  vBlock=1000  

   

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vLap=40  //Start  Dialog  Box  Definition**********************                                                    //Dialog  box  number;                                                          //Control  string  (not  in  dialog  define);                                                                    //Left;  Top;  Width;  Height;  DialogDefine  (99;450;100;300;120;OK!|Enter2HRtn!|Cancel!;"Get  parameters")  DialogAddCounter(99;98;12;    40;  36;15;DisplayNormal!;vBlock;1000;10000;1)  DialogAddText      (99;98;50;    43;250;10;Left!;"Break  seq  into  what  sized  fragments?")  DialogAddCounter(99;98;12;    60;  36;15;DisplayNormal!;vLap;30;100;1)  DialogAddText      (99;98;50;    63;250;10;Left!;"Overlap  between  fragments?")  v99:=  DialogShow  (99;"WordPerfect";;)  v100:=  DialogDestroy(99)  If(v99=2)//Cancel  button  was  hit     Quit()  EndIf       vMaxArray=20000     Declare(vBlockList[1,4])     PosDocBottom  ()     vBot=?GetSelStartEx  ()     PosDocVeryTop()     vTop=?GetSelStartEx  ()     vTick=1     Repeat       If(vTick=1)         vStart=vTop         vEnd=vStart+vMaxArray-­‐1         If(vEnd  >  vBot)           vEnd=vBot         EndIf       Else         vStart=vEnd+1         vEnd=vStart  +  vMaxArray-­‐1         If(vEnd  >  vBot)           vEnd=vBot         EndIf         Declare  vTemp[vTick;4]         vTemp[1..(vTick-­‐1)  ;  1..4]  :=  vBlockList[1..(vTick-­‐1)  ;  1..4]         Discard  (vBlockList[])         Declare(vBlockList[vTick,4])         vBlockList[1..(vTick-­‐1)  ;  1..4]  :=  vTemp[1..(vTick-­‐1)  ;  1..4]         Discard  (vTemp[])       EndIf       vBlockList[vTick,1]=vStart  

   

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    vBlockList[vTick,2]=vEnd       SetSelEx  (vStart;  vEnd)       vBlockList[vTick,3]:=  StrToChars(?SelectedText;Remove!;WhiteSpace!)       SelectOff  ()       vBlockList[vTick,3]:=  StrToChars(vBlockList[vTick,3];Keep!;Alphabetic!)       vBlockList[vTick,4]:=StrLen(vBlockList[vTick,3])       vTick=vTick+1     Until(vEnd=vBot)    FileNew  ()  StyleEditBegin  (Style:  DocStyle!;  Library:  CurrentDoc!)  StyleCodes  (State:  WithoutOffCodes!;  Library:  CurrentDoc!)  Font  (Name:  "Courier  New  ";  Family:  14593;  Attributes:  FontMatchNormal!;  Weight:  90;       Width:  WidthUnknown!;  Source:  DRSFile!;  Type:  TrueType!;  CharacterSet:  FontMatchASCII!)  FontSize  (FontSize:  0.138")  PaperSizeSelect  (Name:  "Letter";  Width:  8.5";  Height:  11.0";  Type:  Standard!;  Orientation:  Landscape!)  SubstructureExit  ()  StyleEditEnd  (State:  Save!)       //Make  real  blocks  with  overlaps     Declare(vFragment[1])     vRow=vBlockList[0;1]//get  number  of  rows  in  first  column  of  array     vRow2=1     For(i;1;i<=vRow;i+1)       If(i<vRow)         vTick=Integer((vBlockList[i,4]-­‐vLap)/(vBlock-­‐vLap))         vLong=((vBlock*vTick)-­‐(vLap*(vTick-­‐1)))         vSeq=StrLeft  (vBlockList[i,3];vLong)         vStartExtra=vLong-­‐vLap+1         vLongExtra=vBlockList[i,4]-­‐vStartExtra+1         vAddExtra=StrRight  (vBlockList[i,3];vLongExtra)         vBlockList[i+1,3]=vAddExtra+vBlockList[i+1,3]         vBlockList[i+1,4]:=StrLen(vBlockList[i+1,3])       Else         vTick=Integer((vBlockList[i,4]-­‐vLap)/(vBlock-­‐vLap))+1         vLong=((vBlock*vTick)-­‐(vLap*(vTick-­‐1)))         vSeq=vBlockList[i,3]       EndIf       For(j;1;j<=vTick;j+1)         If(i=vRow  AND  j=vTick)           Declare  vTemp[vRow2]           vTemp[1..(vRow2-­‐1)]  :=  vFragment[1..(vRow2-­‐1)]  

   

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        Discard  (vFragment[])           Declare(vFragment[vRow2])           vFragment[1..(vRow2-­‐1)]=  vTemp[1..(vRow2-­‐1)]           Discard  (vTemp[])           vFragment[vRow2]=SubStr  (vSeq;vStart;)         Else           If(vRow2=1)             vStart=1             vEnd=vBlock             If(vEnd  >  vLong)               vEnd=vLong             EndIf             vFragment[vRow2]=SubStr  (vSeq;vStart;vBlock)             vStart=vEnd-­‐vLap+1             vEnd=vStart+vBlock-­‐1             vRow2=vRow2+1           Else             If(j=1)               vStart=1               vEnd=vBlock             EndIf             If(vEnd  >  vLong)               vEnd=vLong             EndIf             Declare  vTemp[vRow2]             vTemp[1..(vRow2-­‐1)]  :=  vFragment[1..(vRow2-­‐1)]             Discard  (vFragment[])             Declare(vFragment[vRow2])             vFragment[1..(vRow2-­‐1)]=  vTemp[1..(vRow2-­‐1)]             Discard  (vTemp[])             vFragment[vRow2]=SubStr  (vSeq;vStart;vBlock)             vStart=vEnd-­‐vLap+1             vEnd=vStart+vBlock-­‐1             vRow2=vRow2+1           EndIf         EndIf       EndFor     EndFor       vRow2=vFragment[0]//get  number  of  rows  in  first  column  of  array     For(i;1;i<=vRow2;i+1)       vtoc=RoundOff  ((i/vRow2)*100;0.001)       Prompt  ("Percent  done";"  "+vtoc  ;NoButtons!;150;150)       For(j;1;j<=StrLen(vFragment[i]);j+10)  

   

11    

      Type(SubStr  (vFragment[i];j;10)  +  "  ")       EndFor       HardReturn()       HardReturn()     EndFor  Return    The Oligonucleotide Design Macro was used to generate overlapping oligonucleotide sequences starting from ~1.4 kb dsDNA fragment sequences (with appended vector and restriction site sequences). Application  (WordPerfect;  "WordPerfect";  Default!;  "EN")  Call  (vConstants)  PosDocVeryTop  ()  Type  ("Num  oli  in  each  assm:"  +  vS1N)  HardReturn()  Type  ("Oli  overlap:"+  vS1O)  HardReturn()  Type  ("Length  of  olis:"  +vS1L)  HardReturn()  Type  ("Length  of  oli  assmblies:"  +vS1AL)  HardReturn()  Type  ("Number  of  cassettes:"  +vS2N)  HardReturn()  Type  ("Overlap  twix  cassettes:"  +vS2O)  HardReturn()  Type  ("Length  of  cassettes:"  +vS2L)  HardReturn()  Type  ("Length  of  final  assembly:"  +vS2AL)  HardReturn()  HardReturn()    Type  ("Assembled  cassettes  look  like...")  HardReturn()  HardReturn()    Declare  (vCass[vS2N])  For  (j;1;j<=vS2N;j+1)     If(j=vS2N)       vCass[j]=SubStr(vDoc;vStart;(StrLen  (vDoc))-­‐vStart+1)     Else       vCass[j]=SubStr(vDoc;vStart;vS2L)       vStart=vEnd-­‐vS2O+1       vEnd=vStart+vS2L-­‐1     EndIf     Type(StrLen  (vCass[j]))     HardReturn()  

   

12    

  If(j>=2)       vLeft=StrLeft  (vCass[j];vS2O)       vRight=StrRight  (vCass[j];(StrLen  (vCass[j]))-­‐vS2O+1)       TextColor  (Red:  255;  Green:  0;  Blue:  128)       Type  (vLeft)       TextColor  (Red:  0;  Green:  0;  Blue:  0)       Type  (vRight)     Else       Type(vCass[j])     EndIf     HardReturn()     HardReturn()  EndFor    Type  ("Oligos  grouped  by  cassette,  5'  to  3'  and  ready  to  order...")  HardReturn()  HardReturn()    For  (j;1;j<=vS2N;j+1)     Declare  (vOli[vS1N])     vOliStart=1     vOliEnd=vS1L     For  (i;1;i<=vS1N;i+1)       If(j=vS2N  AND  i=vS1N)         vOli[i]=SubStr(vCass[j];vOliStart;(StrLen  (vCass[j]))-­‐vOliStart+1)       Else         vOli[i]=SubStr(vCass[j];vOliStart;vS1L)         vOliStart=vOliEnd-­‐vS1O+1         vOliEnd=vOliStart+vS1L-­‐1       EndIf       If(i  MOD  2  =  0)         vOli[i]:=  StrTransform  (vOli[i];  "A";  "H")         vOli[i]:=  StrTransform  (vOli[i];  "C";  "I")         vOli[i]:=  StrTransform  (vOli[i];  "G";  "C")         vOli[i]:=  StrTransform  (vOli[i];  "T";  "A")         vOli[i]:=  StrTransform  (vOli[i];  "H";  "T")         vOli[i]:=  StrTransform  (vOli[i];  "I";  "G")         vOli[i]:=  StrTransform  (vOli[i];  "a";  "h")         vOli[i]:=  StrTransform  (vOli[i];  "c";  "i")         vOli[i]:=  StrTransform  (vOli[i];  "g";  "c")         vOli[i]:=  StrTransform  (vOli[i];  "t";  "a")         vOli[i]:=  StrTransform  (vOli[i];  "h";  "t")         vOli[i]:=  StrTransform  (vOli[i];  "i";  "g")         vOli[i]:=  StrReverse  (vOli[i])       EndIf       Type(vOli[i])       HardReturn()     EndFor  

   

13    

  HardReturn()     HardReturn()     Discard  (vOli[])  EndFor  Quit    Label(vConstants)     vDoc:=StrToChars(?GetSelTextAllEx  ();Keep!;Alphabetic!)     vDoc=ToUpper  (vDoc)     vTargetL=  StrLen  (vDoc)     Declare  (vTable[10,9])     vOliMin=54     vOliMax=80     vOliMinNumber=8     vOliMaxNumber=14     vMinCassLap=40     vMaxCassLap=60     vPrompt="PLEASE  PICK  YOUR  CONSTRAINTS"  Repeat  //Start  Dialog  Box  Definition**********************                                                    //Dialog  box  number;                                                          //Control  string  (not  in  dialog  define);                                                                    //Left;  Top;  Width;  Height;  DialogDefine  (99;450;100;150;200;OK!|Enter2HRtn!|Cancel!;"Two  Stage  Oligo  Assembly")  DialogAddGroupBox  (99;98;10;15;  40;  155;  "PLEASE  PICK  YOUR  CONSTRAINTS")  If(Exists(vCheckValues))     DialogAddText  (99;98;10;166;130;10;Center!  |ShadowBox!;"SOME  MIN  IS  LARGER  THAN  A  MAX")  EndIf  DialogAddCounter(99;98;12;    40;  36;15;DisplayNormal!;vOliMin;40;80;2)  DialogAddText      (99;98;50;    43;100;10;Left!;"Min  length  for  an  oligo  (40)")    DialogAddCounter(99;98;12;    55;  36;15;DisplayNormal!;vOliMax;40;80;2)  DialogAddText      (99;98;50;    58;100;10;Left!;"MAX  length  for  an  oligo  (80)")    DialogAddCounter(99;98;12;    85;  36;15;DisplayNormal!;vOliMinNumber;2;24;2)  DialogAddText    (99;98;50;    88;100;10;Left!;"Min  number  of  oligos  (2)")    DialogAddCounter(99;98;12;  100;  36;15;DisplayNormal!;vOliMaxNumber;2;24;2)  DialogAddText    (99;98;50;  103;100;10;Left!;"MAX  number  of  oligos  (24)")    DialogAddCounter(99;98;12;  130;  36;15;DisplayNormal!;vMinCassLap;20;80;1)  DialogAddText    (99;98;50;  133;100;10;Left!;"Min  cassette  overlap  (20)")    DialogAddCounter(99;98;12;  145;  36;15;DisplayNormal!;vMaxCassLap;20;80;1)  DialogAddText    (99;98;50;  148;100;10;Left!;"MAX  cassette  overlap  (80)")    v99:=  DialogShow  (99;"WordPerfect";;)  

   

14    

v100:=  DialogDestroy(99)  If(v99=2)//Cancel  button  was  hit     Quit()  EndIf  If(vOliMin>vOliMax  OR  vOliMinNumber  >vOliMaxNumber  OR  vMinCassLap>vMaxCassLap)     Beep  ()     vCheckValues="Stop"  Else     vCheckValues="Go"  Endif  Until  (vCheckValues="Go")    //End  Dialog  Box  Definition***********************       For  (vS1N;vOliMinNumber;vS1N<=vOliMaxNumber;vS1N+2)       For  (vS1L;vOliMin;vS1L<=vOliMax;vS1L+2)         For  (vS2O;vMinCassLap;vS2O<=vMaxCassLap;vS2O+1)           vS1O=Integer(vS1L/2)           vS1AL=Integer((vS1N*vS1L)-­‐((vS1N-­‐1)*vS1O))           vS2L=vS1AL           If(vS2L<=vS2O)             break           Else             vS2N=Integer((vTargetL-­‐  vS2O)  /  (vS2L-­‐vS2O))             vS2N2=vS2N+1           EndIf           vS2AL=Integer((vS2N*vS2L)-­‐((vS2N-­‐1)*vS2O))           vS2AL2=Integer((vS2N2*vS2L)-­‐((vS2N2-­‐1)*vS2O))           vS2ND  =  Integer(SquareRoot((vTargetL-­‐vS2AL)**2))           vS2ND2=Integer(SquareRoot((vTargetL-­‐vS2AL2)**2))           If(vS2ND2<vS2ND)             vS2N=vS2N2             vS2AL=vS2AL2             vS2ND=vS2ND2           EndIf           For  (i;1;i<=10;i+1)             If(Not  Exists(vTable[i,1]))               vTable[1,1]=vS1N               vTable[1,2]=vS1O               vTable[1,3]=vS1L               vTable[1,4]=vS1AL               vTable[1,5]=vS2N               vTable[1,6]=vS2ND               vTable[1,7]=vS2O               vTable[1,8]=vS2L               vTable[1,9]=vS2AL               Break             Else  

   

15    

            If(vS2ND<=vTable[i,6])                 For  (j;10;j>i;j-­‐1)                   If(Exists(vTable[j-­‐1,1]))                     vTable[j,1]=vTable[j-­‐1,1]                     vTable[j,2]=vTable[j-­‐1,2]                     vTable[j,3]=vTable[j-­‐1,3]                     vTable[j,4]=vTable[j-­‐1,4]                     vTable[j,5]=vTable[j-­‐1,5]                     vTable[j,6]=vTable[j-­‐1,6]                     vTable[j,7]=vTable[j-­‐1,7]                     vTable[j,8]=vTable[j-­‐1,8]                     vTable[j,9]=vTable[j-­‐1,9]                   EndIf                 EndFor                 vTable[i,1]=vS1N                 vTable[i,2]=vS1O                 vTable[i,3]=vS1L                 vTable[i,4]=vS1AL                 vTable[i,5]=vS2N                 vTable[i,6]=vS2ND                 vTable[i,7]=vS2O                 vTable[i,8]=vS2L                 vTable[i,9]=vS2AL                 Break               EndIf             EndIf           EndFor         EndFor       EndFor     EndFor         //Left  Top  Width  Height     DialogDefine  ("vMenu";50;100;1000;160;OK!|Enter2HRtn!|Cancel!;"Oligo  Assembly")     DialogAddText  ("vMenu";"x";200;0;600;13;Center!  |  ShadowBox!;       "PICK  THE  ASSEMBLY  YOU  WOULD  LIKE  TO  USE  TARGET  SIZE:      "  +  vTargetL  +       ":        EXTRA  NUCS  WILL  BE  ADDED  OR  REMOVED  FROM  THE  LAST  OLIGO  IF  NEEDED")     DialogAddGroupBox  ("vMenu";"y";10;15;  30;  120;  "  ")     Declare  (v1[10])     DialogAddRadioButton  ("vMenu";"y";20;21;10;9;"  ";v1[1];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;32;10;9;"  ";v1[2];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;43;10;9;"  ";v1[3];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;54;10;9;"  ";v1[4];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;65;10;9;"  ";v1[5];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;76;10;9;"  ";v1[6];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;87;10;9;"  ";v1[7];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;98;10;9;"  ";v1[8];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;109;10;9;"  ";v1[9];RadioAuto!)     DialogAddRadioButton  ("vMenu";"y";20;120;10;9;"  ";v1[10];RadioAuto!)     For  (i;1;i<=10;i+1)  

   

16    

  If(Exists(vTable[i,1]))       DialogAddText  ("vMenu";"x";50;i*11+10;100;9;Left!;"Num  oli  in  each  assm:"  +  vTable[i,1])       DialogAddText  ("vMenu";"x";160;i*11+10;100;9;Left!;"Oli  overlap:"+  vTable[i,2])       DialogAddText  ("vMenu";"x";270;i*11+10;100;9;Left!;"Length  of  olis:"  +vTable[i,3])       DialogAddText  ("vMenu";"x";380;i*11+10;100;9;Left!;"Length  of  oli  assmblies:"  +vTable[i,4])       DialogAddText  ("vMenu";"x";490;i*11+10;100;9;Left!;"Number  of  cassettes:"  +vTable[i,5])       DialogAddText  ("vMenu";"x";600;i*11+10;100;9;Left!;"Overlap  twix  cassettes:"  +vTable[i,7])       DialogAddText  ("vMenu";"x";710;i*11+10;100;9;Left!;"Length  of  cassettes:"  +vTable[i,8])       DialogAddText  ("vMenu";"x";820;i*11+10;100;9;Left!;"Length  of  final  assembly:"  +vTable[i,9])     EndIf     EndFor     v099:=  DialogShow  ("vMenu";"WordPerfect";;)     v100:=  DialogDestroy(Dialog:  "vMenu")     If(v099=2)//Cancel  button  was  hit       Quit()     EndIf     For  (i;1;i<=10;i+1)       If(v1[i]=1)         vS1N=vTable[i,1]         vS1O=vTable[i,2]         vS1L=vTable[i,3]         vS1AL=vTable[i,4]         vS2N=vTable[i,5]         vS2ND=vTable[i,6]         vS2O=vTable[i,7]         vS2L=vTable[i,8]         vS2AL=vTable[i,9]         Break       EndIf     EndFor     vLong=vTargetL     vStart=1     vEnd=vS2L      FileNew  ()  StyleEditBegin  (Style:  DocStyle!;  Library:  CurrentDoc!)  StyleCodes  (State:  WithoutOffCodes!;  Library:  CurrentDoc!)  Font  (Name:  "Courier  New  ";  Family:  14593;  Attributes:  FontMatchNormal!;  Weight:  90;       Width:  WidthUnknown!;  Source:  DRSFile!;  Type:  TrueType!;  CharacterSet:  FontMatchASCII!)  FontSize  (FontSize:  0.138")  PaperSizeSelect  (Name:  "Letter";  Width:  8.5";  Height:  11.0";  Type:  Standard!;  Orientation:  Landscape!)  SubstructureExit  ()  StyleEditEnd  (State:  Save!)    Return  

   

17    

B. Oligonucleotide pooling. Oligonucleotides were purchased from Integrated DNA Technologies (IDT), resuspended in TE pH 8.0 to a final concentration of 100 µM, and then pooled and diluted to a per-oligo concentration of 25 nM. Upon receiving oligos from IDT in 96 or 384-well plates, plate barcodes were scanned and plates were loaded onto an automated platform (NXp system from Beckman Coulter). Our oligo design manifest file was used to drive the pooling of partitioned oligos into pools of approximately 50 oligos per pool, which constitutes a single dsDNA fragment. We leveraged two styles of automated pooling: (1) pooling via a span-8 style pipetting system in which oligos were re-arrayed in a slower fashion, but with less intervention; and (2) pooling via a 96-well pipetting head in which oligos were instantly pooled once placed into a partitioned reservoir (high-speed with more intervention). C. Single-reaction assembly of dsDNA fragments from overlapping oligonucleotides. Oligo pools were copied into 96-well PCR plates using Beckman Coulter’s NXp system and enzyme master-mixes were dispensed using a bulk reagent dispenser (Preddator from Redd & Whyte). One-step oligo assembly and amplification reactions were setup using an enzyme-master mix consisting of 1X Q5 (NEB) or 1X Phusion (Thermo Fisher) PCR master mix, 0.04% PEG-8000, 500 nM forward and reverse Stage01 PCR primers, and 2.5 nM of the oligonucleotide pool generated above. In general, PCR cycling parameters were 98°C for 2 min, then 30 cycles of 98°C for 30s and 65°C for 6 min (increasing 15 sec/cycle), followed by a single 72°C incubation for 5 min. All thermal-cycling was carried out on Bio-Rad C1000/S1000 cyclers. We analyzed products on 1% E-gels (Invitrogen) alongside a 1kb DNA ladder (NEB) (Figure S12A). In some cases, due to extreme AT content, a 55°C or 60°C annealing/extension temperature was used. In general, 48 oligonucleotides of 60-bases in length were combined to generate ~1.4 kb dsDNA fragments. D. Error correction and re-amplification reactions. PCR reactions from above were cycled at 98°C for 2 min, 2°C/s to 85°C, 85°C for 2 min, 0.1°C/s to 25°C, 25°C for 2 min, and then stored at 4°C. We combined 2.7 µl template DNA with an 8.3 µl error correction mix containing 5.3 µl water, 2 µl Surveyor mismatch-recognition endonuclease (IDT), and 1 µl Exonuclease III (NEB) diluted 1:4000 in water to 25 units/ml. Reactions were then incubated at 42°C for 1 hour. Error corrected templates were then PCR amplified in reactions containing 1X Q5 (NEB) or 1X Phusion (Thermo Fisher) PCR master mix, 500 nM forward and reverse Stage01 PCR primers, and 1:50 error corrected DNA template. In general, PCR cycling parameters were 98°C for 2 min, then 30 cycles of 98°C for 30s and 65°C for 6 min (increasing 15 sec/cycle), followed by a single 72°C incubation for 5 min. We analyzed products on 1% E-gels (Invitrogen) alongside a 1kb DNA ladder (NEB). (Fig S12B). In some cases, due to extreme AT content, a 55°C or 60°C annealing/extension temperature was used to recover synthetic DNA fragments. In general, we observed a 5-10X reduction in error rates compared to untreated samples (41). On average, one error was oberserved per 10,000 bp synthesized (as determined following sequencing of the 7-kb synthetic intermediates below). The 1.4-kb dsDNA fragments were generally produced within 48 hours of oligonucleotide design.

   

18    

E. Assembly, cloning, and MiSeq sequence verification of 7-kb cassettes. Equal amounts (~500 ng) of stage 1 error-corrected PCR fragments were combined 4- or 5- at-a time. One-fifth volume of NotI restriction enzyme (NEB) was added and the reactions were carried out at 37°C for one hour. We then processed and concentrated the reactions with a PCR cleanup kit (Qiagen). Reactions were then separated on a 1% agarose gel and fragment pools were gel extracted and purified (Qiagen). The overlapping fragments were then simultaneously assembled into a PCR-amplified pCC1BAC cloning vector using the the Gibson Assembly® HiFi 1-step kit (SGI-DNA) and transformed into Epi300 electrocompetent E. coli cells (Illumina) as previously described (42, 43). Twenty-four colonies from each 1st stage cassettes were picked from petri plates using an automated colony picking system (QPix system from Molecular Devices). Picking this number of colonies ensured that we were able to identify error-free cassettes during this initial pass thus reducing the need to recirculate back through the process. Bacterial colonies were formatted within a deep-well growth block in such a way that dozens of 1st stage cassettes could be “collapsed” into a single group of 24 wells via Beckman Coulter’s NXp (96-well head) after 20 hours of growth in a shaking incubator (Figure S13). Formatting in this fashion allowed us to screen many clones without having to increase next-generation sequencing libraries construction throughput by 10X. This single grouping of 24 wells was plasmid prepped using Agencourt CosMCPrep (Beckman Coulter) on an automated platform (NXp system from Beckman Coulter), and the resulting plasmid DNA was used to create 24 indexed libraries using Illumina’s Nextera XT system per the manufacturer’s protocol. Samples were then sequenced on Illumina’s MiSeq platform using reagent kit V2 and a 2x150 bp run type. Illumina’s Nextera process was also automated by using Eppendorf’s epMotion 5073 and Alpaqua’s LE Magnet Plate for low volume bead elution. After MiSeq sequencing was complete we then initiated our Clone Verification Analysis (CVA) pipeline to identify and select error-free cassettes. A manifest file describing a library-reference association matrix was filled out prior to launch of analysis. The sequenced libraries were quality-trimmed using Trimmomatic 0.32. Concurrently, we created a comprehensive reference sequence by inserting the expected cassette sequence at insertion sites of the vector used. The reference indexes were built using bowtie2-build. Mapping of each library was then performed using bowtie2 with default alignment parameters against all appropriate references described in the manifest file. The variants in the library were detected by analyzing each BAM file using samtools mpileup. The result was then filtered using bcftools’ varfilter and saved as a VCF file. Finally, the VCF files were summarized into a single table that would allow quick identification of the error-free cassettes. Furthermore, this output file was used to drive the automated selection of the cultured clones by using Beckman Coulter’s NXp platform (span-8).

E. coli clones containing error-free 7-kb cassettes were usually obtained within one week of oligonucleotide design.

F. Assembly of overlapping DNA fragments in yeast. Error-free cassettes were prepared from 10-ml induced E. coli cultures and inserts were released by digestion with the AsiSI restriction enzyme (NEB). Equal amounts (~500 ng) were combined as many as 15- at-a time and then processed and concentrated with a PCR cleanup kit (Qiagen).

   

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Between 50-250 ng of each fragment were combined with 50 ng EVW vector and transformed into yeast as previously described (7, 10, 44, 45). Yeast clones were first screened by multiplex PCR at the assembly junctions and then by separation of supercoiled DNA on agarose gels alongside a supercoil ladder (see below).

Yeast clones containing 1/8th molecules were usually obtained within two weeks of oligonucleotide design. G. Plasmid DNA isolation from yeast. Yeast centromeric plasmid (YCp) DNA was prepared as follows (See also (10, 44)). A 5-10 ml S. cerevisiae culture was grown overnight at 30oC in complete minimal (CM) medium minus tryptophan (Teknova). Cells were centrifuged and resuspended in 250 µl of buffer P1 (Qiagen), containing 5 µl of Zymolyase-100T solution (10 mg/ml zymolyase-100T [US Biological cat. no. Z1004], 50% (w/v) glycerol, 2.5% (w/v) glucose, 50 mM Tris·Cl, pH 7.5). Following an incubation at 37oC for 1 hour, 250 µl of lysis buffer P2 (Qiagen) were added. Tubes were inverted several times and incubated at room temperature for 5 min. Then, 250 µl cold neutralization buffer P3 (Qiagen) were added, and the tubes were inverted several times, and the samples were microcentrifuged for 10 min at 16,500 × g. The supernatant was transferred into a fresh tube and precipitated with 700 µl isopropanol followed by a 70% ethanol wash. The DNA pellet was resuspended in 30-50 µl TE buffer, pH 8.0. To estimate the size of the purified YCp DNA, 10 µl of the plasmid preparation were separated on a 1% agarose gel in 1xTAE buffer (No ethidium bromide) by constant voltage (3hr at 4.5V cm-1). After the electrophoresis the gel was stained with SYBR Gold and scanned with a Typhoon 9410 imager (GE Healthcare Life Sciences). H. Rolling Circle Amplification (RCA) reactions. MDA reactions were generally performed using the TempliPhiTM Large Construct DNA Amplification kit (GE Healthcare), which generated an error approximately once per 50,000 bp synthesized (as determined by cloning and sequencing individual molecules. Briefly, 4 µl of yeast plasmid preparation (above) were added to 9 µl of sample buffer. The mixture was incubated at 95oC for 3 min and then placed on ice. Ten microliters of reaction buffer and 0.5 µl of the enzyme were added to the denatured sample mixture. The amplification reactions were incubated at 30oC for 16-18 hours. The enzyme was inactivated at 65oC for 10 min. Five microliters of amplified DNA were digested with the restriction enzyme NotI in 50 µl volume at 37oC for one hour. Twenty microliters of the digest were separated on a 1% agarose gel with EtBr in 1xTAE buffer by FIGE/U9 electrophoresis. In some cases, the REPLI-g mini kit (Qiagen) was used for the amplification following the manufacturer’s instructions for purified genomic DNA. Five microliters of yeast plasmid preparation were used as template in an amplification reaction of 50 µl. Reactions were incubated overnight at 30oC. Five to ten microliters of amplified DNA were digested with the restriction enzyme NotI in 50 µl volume at 37oC for one hour. Twenty microliters of the digest were separated on a 1% agarose gel with EtBr in 1xTAE buffer by FIGE/U9 electrophoresis (Figure S14, (7)). I. Cassette manipulations. In some cases, sequence-verified cassettes from HMG, RGD1, and RGD2 were further manipulated to match the present design. Figure S16 illustrates how this was performed. Cassettes were PCR amplified upstream and

   

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downstream of a site of insertion or deletion. Genes to be added back were amplified using JCVI-Syn1.0 genomic DNA as template. To make a base substitution, the change is made within the PCR primer. Vector and insert DNA fragments were designed such that they contain 40bp overlaps to facilitate in vitro DNA assembly. Newly assembled cassettes were sequence verified following cloning, as described above, prior to assembly in yeast to generate the new version of the respective 1/8th molecule. J. PacBio complete genome de novo assemblies. An alkaline-lysis approach followed by phenol extraction and ethanol precipitation was used to isolate high molecular weight DNA from Syn3.0 transplants. DNA was quantitated (Qubit, Thermo Fisher Scientific) and quality controlled using an E-Gel (Thermo Fisher Scientific) and then purified using AMPure PB (Pacific Biosciences). The samples (approximately 8-10 µg) were then sheared to an average of 8-20 kb using a g-TUBE (Covaris) at 4500 RPM in an Eppendorf 5424 centrifuge. The samples were then cleaned (Power Clean Pro DNA Clean-Up kit, Mo Bio), quantitated (Qubit, Thermo Fisher Scientific) and quality controlled (Bioanalyzer, Agilent). Adhering to the Pacific Biosciences template preparation protocol (SMRTbell Template Prep Kit 1.0), the samples were first treated with Exo VII to remove single-stranded ends from the DNA fragments, and then taken through DNA damage and end repair before being re-purified using AMPure PB beads. SMRTbell adapter ligation was then performed overnight and failed ligation products were removed with Exo III and Exo VII. AMPure purified DNA was again quantitated (Qubit, Thermo Fisher Scientific) and quality controlled (Bioanalyzer, Agilent) before being size selected (2 or 8 kb to 50 kb) using the BluePippin (0.75%, DF Marker S1 high pass 6-10 kb v3, Sage Science). The size selection was then AMPure PB purified and verified on the Bioanalyzer (Agilent). Sequencing primer was then annealed to the size-selected SMRTbell templates followed by polymerase binding (DNA/Polymerase Binding Kit P6 v2). The prepared libraries were then bound to magnetic beads and loaded onto the Pac Bio RS II at a concentration of 0.200 nM (DNA Sequencing Kit 4.0, Pacific Biosciences). Between 210 and 760 MB of data was generated using one SMRT Cell (V3) per library. Reads of insert ranged from 3700 bp and 9500 bp with polymerase lengths ranging from 11,000 bp to 15,000 bp.

Each sample was assembled de-novo using SMRT Analysis 2.3.0 RS_HGAP_Assembly.3 protocol. In short, subreads were extracted using the standard SMRT Analysis 2.3.0 P_Filter protocol using readScore = 0.75 and minSubReadLength = 500 yielding between 709 and 1151 Mbp of filtered subreads per sample with mean subread lengths between 3475 and 8427 bp. Subreads were then error corrected using the P_PreAssemblerDagcon module using computeLengthCutoff = True and genomeSize = 600000 yielding between 6.3 and 9.9 Mbp error corrected reads per sample with N50 lengths between 8780 and 24344 bp. Error corrected reads were assembled to unitigs using the P_AssembleUnitig module and polished using the P_AssemblyPolishing module both using default parameters. The assembly resulted in a single circular contig matching the expected syn3.0 reference size. Overlapping regions on the 5’ and 3’ end of the circular contigs were later manually trimmed using CLC Genomics Workbench, finalizing the complete genome. In order to identify the differences between the expected and assembled genomes, each assembled reference genome was mapped to its corresponding expected reference using BWA-mem Version: 0.7.12-r1039 with default settings.

   

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Variants were then called with CLC Genomics Workbench 8.0.2 using the Basic Variant Detection tool with "Minimum coverage = 1" and "Minimum count = 1”, resulting in 9 to 27 variant calls per sample. These variants were confirmed using Illumina MiSeq 2x250bp reads (processed as described above).

Methods to analyze growth characteristics of transplant cells A. Growth conditions and colony purification. To characterize growth properties of JCVI-syn1.0 and derivative transplant strains with reduced genomes, cultures were grown at 37°C in SP-4 liquid medium (containing 17% fetal bovine serum) or on solid medium of the same composition, supplemented with 1% agarose. Initial transplant colonies obtained under selection were picked and propagated in SP4 liquid medium without selection. Static liquid cultures were mixed by trituration and passed through 0.2-µm syringe filters (Acrodisc®, Pall Life Sciences) with gentle pressure. The filtrate was immediately diluted in SP4 medium and 10-fold dilutions were plated on solid SP4 agarose medium. Well-separated colonies from near-limit dilutions were imaged for comparison of size with a stereomicroscope (SZM-45T2, AmScope) and picked for subsequent growth and molecular genetic or phenotypic characterization. Notably, all populations analyzed were filter cloned by this procedure and ultimately were propagated from replicative units that passed through 0.2-µm filters. B. Measurement of growth rates. To avoid factors that can confound both the measurement of mycoplasma cell growth and the comparison of cells with altered genome content (e.g. differences in the mode of replication, physical aggregation, rates of cell death, altered metabolic indicators, or interference by serum proteins in growth medium), we developed a method (13, 46) to compare replication rates by a direct measure of cell-associated nucleic acid. Specifically, we used the fluorescent stain Quant-iT™ PicoGreen® (Molecular Probes®, Invitrogen™) which binds dsDNA (and to a far lesser extent dsRNA) to quantify the rate of increase during logarithmic-phase cultures in liquid medium. To measure logarithmic growth rates, mycoplasma transplants were grown in static, planktonic culture at 37°C in SP4 liquid medium (without tetracycline or Xgal). Overnight late-logarithmic phase cultures were diluted approximately 500-fold with pre-warmed medium and distributed in replicate 0.80-mL aliquots into graduated 1.7-mL microcentrifuge tubes. Individual tubes were removed at selected times and placed on wet ice to arrest growth. To obtain cells without material loss or contaminating medium components, the collected culture aliquots (or controls containing only medium) were underlain in situ with 0.40-mL sucrose cushions (0.5 M sucrose, 20 mM Tris HCl; pH 7.5) and cells were sedimented by centrifugation at 16,000 x g for 10 min. The top layer of medium and cushion were removed by vacuum aspiration and the remaining clear cushion was further adjusted to 100-µL without disrupting pellets. Cells were lysed by adding 50 µL of 0.3% (w/v) SDS in TE, pH7.5 (final concentration 0.1%) followed by trituration and incubation at 37°C for 5 min. Lysates were diluted to 0.01% SDS by adding 1.35 mL of

   

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TE, and mixed by rotary inversion for 1 hr at room temperature. To quantify nucleic acid, equal volumes (80 µL) of diluted lysate and Quant-iT™ PicoGreen® reagent (prepared as described by the manufacturer) were mixed in wells of opaque black 96-well plates (Costar, cat. 3915) and incubated in the dark at room temperature for 5 min. Fluorescence was measured using a FlexStation 3 fluorimeter (Molecular Devices) with excitation at 488 nm, emission collected at 525 nm, and a cutoff setting of 515 nm. The net relative fluorescence units (RFU) of samples (after subtraction of RFU from medium control lacking cells), were plotted as log2 (RFU) vs. time (min) and the doubling times were calculated from the slopes of exponential regression curves (R2, Figure S17) using the formula: doubling time=ln2/exponential rate. Exponential curves generated from cultures diluted 2-fold with complete medium prior to sample processing demonstrated a high correlation between log2 RFU and cell concentration, over a RFU range of approximately 64-fold (Figure S17). Linear regions of semi-logarithmic plots within this range were used to calculate exponential replication rates from growing cultures. The accuracy and reproducibility of the technique (reflected in R2 values) allowed the use of single samples. To avoid minor variables such as batch differences among medium preparations and temperature fluctuations, constructs were compared under identical conditions and within a single experiment. C. Light Microscopy. To observe natural cell morphologies in static cultures without manipulation, wet mounts in medium were prepared by depositing 3µL of settled cells, carefully removed by micro pipette tip from round-bottom culture tubes, onto an untreated glass slide and applying a 18 x 18 mm cover slip. Light microscopy was performed using a Zeiss Axio Imager 1 microscope with a Zeiss plan/apochromatic 63x oil 1.4 objective and differential interference contrast (DIC) optics. D. Scanning Electron Microscopy. Cells grown in SP4 medium were centrifuged at room temperature for 4  min at 2,000xg to produce a loose pellet. Medium (950  µl) was removed and replaced with 1  ml of fixative. The fixative solution was 2.5% glutaraldehyde, 100  mM sodium cacodylate, 2  mM calcium chloride and 2% sucrose (fixative was added cold and samples were stored at 4  °C). Cells were immobilized on polyethylenimine or poly-D-lysine coated ITO glass coverslips for 2  min and washed in 0.1  M cacodylate buffer with 2  mM calcium chloride and 2% sucrose for 5 × 2  min on ice. Cells were post fixed in 2% osmium tetroxide with 2% sucrose in 0.1  M cacodylate for 30  min on ice. Cells were rinsed in double distilled water and dehydrated in an ethanol series (20, 50, 70, and 100%) for 2  min each on ice. Samples were critical point dried (with CO2) and sputter-coated with a thin layer of Au/Pd. Samples were imaged with a Zeiss Merlin Fe-SEM at 2.5  kev, 83  pA probe current and 2.9  mm working distance (zero tilt) using the in-lens SE detector.

Miscellaneous methods A. Yeast strains, growth condition, and genetic engineering. The yeast Saccharomyces cerevisiae (S. cerevisiae) strains used here were VL6-48 (MATa, his3Δ200, trp1Δ1, ura3-52, lys2, ade2–101, met14) containing the JCVI syn1.0 (10) or syn1.0/∆RE∆IS genome (39), and VL6-48 (MATa, his3Δ200, trp1Δ1, ura3-52, lys2, ade2–

   

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101, met14). Yeast cells were grown in standard rich medium containing glucose (YEPD) or galactose (YEPG); or in synthetic defined (SC) minimal dropout medium (38). For the URA3/5-FOA counter-selection (47) cells were grown on SC- HIS, supplemented with 5-fluoroorotic acid (5-FOA) (1 mg/ml). For kanamycin selection, cells were grown on YEPD, supplemented with 200 µg/ml of Geneticin G418 (Cat#: 11811-023, Life technologies). Yeast transformation was carried out by either Lithium –acetate (48) or spheroplast (49) methods. A number of genetic tools were used to perform gene(s) deletion and insertion in a mycoides genome cloned in yeast. These include (a) the TREC method (38) for scarless gene cluster deletions in the D-series genomes (Table S6) and in generation of restriction NotI sites (GCGGCCGC) in 8 NotI strains, (b) the TREC-IN method (50) for scarless gene knock-in and knock-out, (c) insertion of the MET14 marker or a landing pad cassette (18), and (d) gene or gene cluster deletion by the TRP1 marker and the CORE6 cassette (38). All cassettes or markers for yeast transformation were generated by PCR using chimeric primers to introduce additional 40 to 50 bp to ends of PCR products for homologous recombination to upstream and downstream of target site. All primers, listed in the tables, were purchased from IDT (Integrated DNA Technologies). The Advantage HD Polymerase (Clontech), unless otherwise indicated, was used for polymerase chain reaction (PCR). In general, PCR was performed for 30 cycles. In some cases, if two rounds of PCR were involved, the first round was performed for 18 cycles and the second round was performed for 22 cycles using the first round of PCR product as DNA template. The PCR product was, if needed, purified by the MinElute PCR Purification Kit (Qiagen) according the manufacturer’s instruction. Approximately 0.5 to 1 µg of PCR product was used for yeast transformation. Correct integration was screened by junction PCR, unless otherwise indicated, to detect the presence of 2 junctions between an insertion marker (or a cassette), and upstream and downstream of target region. In general, at least two positive clones of all engineered mycoides genomes were chosen for genome transplantation. B. Engineering NotI sites into the genome. Generation of a NotI site was performed by the TREC method (38). To engineer a NotI site, 5 to 9 nucleotides (lowercase in primer sequence in the Table S12) were embedded into the primers used for PCR-amplifying the CORE cassette. If a NotI site was generated within a coding region, a reading frame was maintained by inserting either 9 bp or 6 bp able to generate a NotI site together with adjacent nucleotides. The production of the CORE cassette was generated by two rounds of PCR. Primers used for PCR, diagnosis, and the location of all NotI sites on the genome are shown in Table S12. Since a non-essential region (24.9 kb) between the 1/8th segments 1 and 8 was not included in the 8- NotI -segments design (Figure S9A), 208 bp which contains 8 bp of NotI site and a 200 bp overlapping to the adjacent segment 1 was inserted into the location of NotI -8D. Using the JCVI1.0-Syn1.0 genome DNA as template, a 305-bp fragment was PCR-amplified by a pair of chimeric primers listed in Table S12. The 50-bp of the 5’ PCR product is for homologous recombination to the target site and the 50-bp of the 3’ PCR product is overlapped with the 5’ end of a CORE cassette amplified by another chimeric primer. Approximately 0.5 µg of two purified PCR products were co-transformed to yeast. Correct insertion was screened by junction PCR. The procedure of recycling of the CORE cassette was same as that in the D serial strains.

   

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C. Insertion of MET14 into syn1.0 segment 6. The yeast MET14 marker was PCR-amplified by primer pair 5’AGTATAAAACTTTTATCCTAACCGATTTTAAGTTTTATACAGGAGGAATTGCACCCACGGTACCTTAC-3’ and 5’CCAACAATGTTTTATCTTATTTAAAATATTAATAGGTTGGAGGATTAAAAGGTCTATACTATAATACATCAAC-3’ using Yeast W303 genomic DNA as template and the Ex Taq DNA Polymerase (TaKaRa), according to the manufacturer’s instructions. The PCR product was transformed into NotI strain 6 and selected on synthetic defined (SC) minimal dropout medium (38) SC-MET plates. The correct MET14 marker replacement was screened by colony PCR using the primer pair 5’- TCAACATATTCTGGTATGTCT-3 and 5’- ATATTCTACCGGTTTTTCTACC-3’ flanking the upstream and downstream target region. D. Constructions of 8 landing pad strains. PCR amplification was used to produce a unique landing pad cassette (2,250 bp) that allowed individual replacement of each 1/8th segment into the JCVI syn1.0 genome by homologous recombination in yeast. Seven landing pad cassettes were PCR-amplified using plasmid pRC72 (unpublished) as the DNA template to replace segments from 2 to 8. One landing pad cassette was PCR-amplified using plasmid pRC73 (unpublished) as a DNA template to replace segment 1, which contains a yeast centromeric plasmid (YCp). The primers for PCR are listed in Table S13. After transformation, cells were selected on SC-MET, transformants with correct replacement of the cassette were screened by junction PCR. The landing pad insertion genome was further confirmed by contour-clamped homogeneous electric field CHEF gel electrophoresis. E. Construction of single-reduced segment genomes (RGDs) by RMCE (18). To build a single RGD segment genome, 1/8th RGD and 7/8th JCVI syn1.0 were brought together in a diploid strain (Fig S10). A landing pad strain containing the MET14 marker was first crossed with a corresponding opposite mating type yeast strain that contains a 1/8th RGD segment carried by a donor vector with the TRP1 marker. It was done by mixing two opposite strains on a yeast extract peptone dextrose (YEPD) plate and incubating the plate overnight at 30oC. Diploid strains were selected on a SC- TRP-MET plate. The expression of Cre recombinase was induced by growing diploid cells on YEP-galactose plate for two days. The event of cassette exchange was screened by the restoration of uracil prototrophy. The cells from the YEPG were replica plated on SC-URA for 2 to 3 days until colonies appeared. The structure of the semi-RGD genome was first verified by PCR analysis for the boundaries between the RGD segment and the JCVI syn1.0 genome. The PCR products were further digested with restriction enzyme NotI to confirm 1/8th RGD segment is flanked with NotI site as design. Multiple positive clones were chosen for transplantation. Generally, colonies could be seen in 3 to 4 days after genome transplantation, except transplanted cells from the RGD1.0-6 semi-genome which required about 10 days to give a colony similar in size to the others (See Fig 18S to 21S). All 1/8th RGD segments in semi-genomes could be released by NotI restriction enzyme and purified for genome assembly.

   

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F. Modification of RGD segment 6 by insertion of a landing pad. Two versions of RGD segment 6 (RGD1.0-6sf and RGD1.0-6P) were further modified by inserting a special landing pad (LP) cassette to produce RGD1.0-6sf-LP and RGD1.0-6P-LP, respectively. The first one was the mycoides strain that spontaneously self-fixed the expression of gene MMSYN1_0621 and the second was a redesigned segment 6 that contains a corrected promotor for gene 0621. A modified landing pad was generated by 2 rounds of PCR. The first round PCR was performed by the primer pair 5’TTTAAAATATAAAATAAAAATAAAGGAAAAAATATTAGAAGGGAATATTAGCACCCACGGTACCTTAC and 5’ATTCAGTACCGTTCGTATAGCATACATTATACGAAGTTATAGGGTAATAACTGATATAATTAAATTG using a mycoides genome with the insertion of the modified landing pad (unpublished) as DNA template. The second PCR was carried out by primer pair 5’TTTAAAATATAAAATAAAAATAAAGGAAAAAATATTAGAAGGGAATATTAGCACCCACGGTACCTTAC And 5’TTGAGGATATTGAGGACATTCTTCTAATATAGCTGTTAATTCTGATTCAGTACCGTTCGTATAGC-3’) using the first round PCR product as template. The PCR product was transformed into yeast containing a single-RGD segment genome with either the RGD1.0-6sf or RGD1.0-6P. The cassette was inserted between genes 0601 and 0606 in RGD segment 6. A correct insertion was detected by screening the junctions using 2 primer sets: 5’-ATATGACAAAATCCAAGACAAC-3’ and 5’-ACGTGAAATTAGTGGCACG-3’) and (5’-ACCGTGGATGATGTGGTC-3’ and 5’-ATGGGTTTTCTTCCTAACTG-3’). Two modified RGD genomes were transplanted to produce cells containing either RGD1.0-6sf-LP or RGD1.0-6P-LP. G. Construction of RGD1-3s by adding a gene cluster to RGD1-3. The gene cluster (MMSYN1_0217 to 0219) was seamlessly inserted back into its original locus by the TREC-IN method (18). It was done by 2 rounds of transformation. The CORE6 cassette was first PCR-amplified using the plasmid pCORE6 as DNA template and primer pair 5’ATCAATTGATGAGCTTTTCTTTTGCTTAATTTATTAAGTTATAAAATTTAGGGATAACAGGGTAATACGGATTAG-3’ and 5’TTTTTTCAAGTAATTCTTATAAAATTTTGAATTTTTTAAAAAAGAATATTGGCCAGCCATTACGCTCG-3’. About 0.5 to 1 µg of the purified PCR product was transformed into yeast harboring the semi-RGD genome containing the 1/8th RGD1-3 segment and selected on SC-URA plates. After junction PCR screening, a positive clone was subjected to the second round of transformation to insert the 3-gene cluster (3.6 kb) by a knock-in module, consisting of the 3’ KanMX4 gene, a 50-bp repeat sequence, and the cluster. The 3’ KanMX4 gene was PCR-amplified for 18 cycles using the pFA6a-kanMX4 as template and a pair of primers 5’CTGATGATGCATGGTTACTC -3’ And

   

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5’TAAATTAAGCAAAAGAAAAGCTCATCAATTGATGACAGTATAGCGACCAGCATTC-3’. The second round of PCR was performed for 22 cycles using the first round PCR product as DNA template and a pair of primers (5’- CTGATGATGCATGGTTACTC -3’ and 5’-AGCAATGGCTGTAAAATAAAATTTTATAACTTAATAAATTAAGCAAAAGAAAAGCTCA-3’). The cluster was PCR-amplified using the JCVI-syn1.0 genome as template and a pair of primers 5’TCTTTTGCTTAATTTATTAAGTTATAAAATTTTATTTTACAGCCATTGCTATTAATC-3’ and 5’TTTTTTCAAGTAATTCTTATAAAATTTTGAATTTTTTAAAAAAGAATATTATGAAGCAAACAAAAGTTGATATTTG-3’. The second step was performed by co-transformation of these two PCR products with a 50-bp overlapping sequence (shown in bold). The 3-gene cluster recombined with the 3’Kan gene was inserted into the target locus by selection for kanamycin resistance. After transformation, cells were incubated in YEPD liquid medium overnight at 30oC, followed by growing on YEPD plates supplemented with Geneticin G418. Precise integration was screened by PCR. The procedure of recycling the cassette to produce a scarless insertion was identical to that for the TREC cassette described in the TREC method (38). The viability of the modified genome was tested by transplantation. H. Purification of 1/8th genome segments. All 1/8th genome segments were isolated from M. mycoides genomes. Cells were grown in 10 ml SP4 medium containing tetracycline-resistance (tetM) overnight at 37°C. Cells were harvested by centrifugation at 4,575g for 15 min at 10°C. Cell pellets were washed with 5 ml of washing buffer (10 mM Tris and 0.5 M sucrose, pH 6.5) and harvested again as before. Cells were re-suspended in 100 µl of washing buffer and incubated at 50°C for 5 min. Cell suspensions were mixed with 120 µl of 2% agarose (Cat#: 16500-500, Invitrogen) in 1× TAE buffer (40 mM Tris-acetate and 1 mM EDTA), pre-warmed to 50°C. Approximately 100 µl of this mixture was added to agarose plug molds (Cat# 170-3713 Bio-Rad). After setting 30 min at room temperature, plugs were lysed and proteins were digested with Proteinase-K solution [500 µl of the Proteinase-K buffer (Bio-Rad) and 20 µl of Proteainase K (>600 mAU/ml) (Cat# 19133, Qiagen)] at 50°C overnight. Agarose plugs were washed once with 1 ml of 0.1X Wash Buffer (Bio-Rad CHEF Genomic DNA Plug Kit) for 30 to 60 min, followed by washing with the same buffer containing 0.5 mM PMSF (phenylmethylsulfonyl fluoride) for 30 min. Plugs were equilibrated with 1ml of 1X Buffer 3 (NEB) for 30 min twice. The plug was treated with 50 units of NotI for 5 hours at 37oC in 250 µl 1X buffer 3 containing bovine serum albumin BSA (NEB) in 100 µg/ml concentration. After digestion, the plug was loaded on a 1% agarose gel and subjected to Field-Inversion Gel Electrophoresis (FIGE, BioRad, catalog # 161-3016) in 1X TAE buffer. The parameters of the electrophoresis were forward 90 V, initial switch 0.1 sec, final switch 10 sec, with linear ramp, and reverse 60 V, initial switch 0.1 sec, final switch 10 sec, with linear ramp for 16 hours at room temperature. The gel was then stained with 1X TAE buffer containing ethidium bromide (0.5 µg/ml) for 30 minutes. To elute 1/8th genome segments, a REC0-CHIP membrane filter (Takara, code# 9039) was inserted next to the band. The gel was re-orientated by 90° so that the DNA could be run into the membrane. The gel was subjected

   

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to electrophoresis for 2 hours at 3.5 Volt/cm. The RECO-CHIP filter was placed into the RECO-CHIP DNA collection tube and spun for 2 min at 500X g. In general, about 20 to 30 µl DNA solution was collected and kept at 4°C. 1 to 2 µl of purified DNA was analyzed on a 1% agarose gel by FIGE using the same conditions described above. I. Genome assembly by the yeast method. All 1/8th segments of RGD and syn1.0 segments used for genome assembly are listed in Table S7A. Genome assembly was carried out in the yeast VL6-48 strain. The spheroplast transformation procedure (8) was used with minor modifications as follows. Yeast cultures were harvested at an OD600 ≈ 2. After centrifugation, cell pellets were re-suspended in 20 ml of 1M sorbitol solution and kept at 4oC for 4 to 20 hours. Approximately 20 to 50 ng of each of the 1/8th genome segments were mixed in a final volume of 50 µl and then added to about 1 X 108 yeast spheroplasts. After transformation, yeast spheroplasts were regenerated and selected on a sorbitol-containing (1) SC-HIS, (2) SC-HIS-TRP, or (3) SC-HIS-TRP-MET. J. Yeast DNA preparation for PCR. Yeast cells were patched to an appropriate selection medium and grown overnight at 30oC. Cells (visible amount equal to approximately 1 µl of cell mass) were then picked by pipette tip and twirled in 0.5 ml of a PCR tube containing 10 µl of the zymolyase solution [10 µl of sterile water + 0.5 µl of 10 mg/ml of zymolyase 20T (ICN Biochemicals)]. The tube was incubated at 37oC for 1 hour, followed by 15 min incubation at 98oC. 1 µl of zymolyase-treated cells was analyzed by PCR in 10 µl reaction volume using the QIAGEN Fast Cycling PCR Kit (cat# 203741, Qiagen), according to the manufacturer's instructions. About 1/3rd of the PCR product was analyzed on a 2% E-gel (Invitrogen) using the E-Gel Power Base Version 4 (Cat# G6200-04, Invitrogen) for 30 min. K. Multiplex PCR screening to confirm genome assemblies. To screen for complete genome assembly, multiplex PCR was performed by QIAGEN Multiplex PCR Kit (cat# 206143, Qiagen) using a unique set of primer mixes, each of which contains 8 primer pairs, listed in Table S15. DNA prepared from yeast for PCR was described above. A 15 µl PCR reaction contained 1 µl of zymolase-treated yeast cell suspension, 1.5 µl of 10X primer mix, 6 µl of PCR-grade water, and 7.5 µl of the 2X master mix. The PCR conditions were 94 °C for 15 min, then 35 cycles of 94 °C for 30 s, 52 °C for 90 s, and 68 °C for 2 min, followed by 5 min at 68 °C for one cycle. 5 µl of PCR product was analyzed on a 2% E-gel for 30 min. The primer mixes for different multiplex sets are listed in Table S15. L. Deletions of 39 gene clusters and single genes in the RGD24*678 genome. The plasmid pCORE6 was used as DNA template to amplify the CORE6 cassette for the deletions. Two rounds of PCR were performed using primer sets listed in the Table S14. Yeast transformations were selected on SC-URA and correct deletions were screened by PCR to detect insertion junctions. Viability of each deletion was examined by transplantation. M. Gene cluster deletions and gene 0154 insertion in clone 48. A 2-cluster deletion (∆33∆36) in clone 48 genome was used to create the JCVI-syn2.0 genome. To remove the

   

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CORE6 cassette, the recycling construct consisting of the 3’ truncated KanMX4 gene and a 50 bp repeat sequence was produced by 2 rounds of PCR amplification. The 3’ KanMX4 gene was PCR-amplified for 18 cycles using pFA6a-kanMX4 as template and a pair of primers 5’CTGATGATGCATGGTTACTC3’ and 5’TATGTTCCAATCTTAAAATCTATTTTAATAATATCAAAACATTACTAACAGTATAGCGACCAGCATTC3’. The second round of PCR was performed using the first round PCR product as DNA template and a pair of primers 5’CTGATGATGCATGGTTACTC 3’ and 5’ATGCAGGTTTTCCTCCTCTACACCTGCATTTTTTATTTAAAATATGTTCCAATCTTAAAATCTATTTT3’.

After transformation, cells were selected on Geneticin G418 plates as described in section G above. Correct insertions were screened by junction PCR using the primer pair 5’CAGAAAGTAATATCATGCGTC3’ and 5’TTTACACAAAACAATTGACTCAG3’.

The procedure for removal of the cassette was described above. The resulting genome was subjected to gene knock-in by the TREC-IN method ((50) and Fig S11). The CORE6 cassette was generated by 2 rounds of PCR amplification using pCORE6 as DNA template and 2 primer sets. 1st set: 5’ATACTTATATGAGGTGAGATTTAGGGATAACAGGGTAATACGGATTAG3’ and 5’- AGATATAAAAAGGAAGAGATGGCCAGCCATTACGCTCG-3’; 2nd set: 5’ATTTAGTTACTTTTAAATATTTTTTTCTAATACTTATATGAGGTGAGATTTAGand 5’TGTTTTATGTTATAAAAAAGTTAGTAACATAGATATAAAAAGGAAGAGATGGC3’.

After transformation, cells were selected on SC-URA. A correct integration was verified by junction PCR using 2 primer sets L junction: 5’-AGAAGCTAGAAAACGTGAATAC and 5’-CTTCGGAGGGCTGTCACC, R junction: 5’-GTTGCATTCGATTCCTGTTTG and 5’-AATGCATGCTTATAATGAGTATG.

Positive clones were subjected to a second round of transformation to insert gene 0154. The strategy of gene insertion is the same as the insertion of gene cluster 0217-0219 described above (section G). The 3’ KanMX4 gene was PCR-amplified in 2 rounds using 2 sets of primer pairs Pair-1: 5’-CTGATGATGCATGGTTACTC-3’ and 5’CTCACCTCATATAAGTATTAGAAAAAAATATTTAAAACAGTATAGCGACCAGCATT-3’ and Pair-2: 5’-CTGATGATGCATGGTTACTC-3’ and 5’ATTGTTCAGTTTTTAATTATTAATCTCACCTCATATAAGTATTAGAAA-3’)

   

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Gene 0154 was PCR-amplified using the syn1.0 genome as DNA template and the primer pair 5’TTTTTCTAATACTTATATGAGGTGAGATTAATAATTAAAAACTGAACAATTAGTTTC-3’ and 5’TATGTTATAAAAAAGTTAGTAACATAGATATAAAAAGGAAGAGATGTTCAAGAATTTATTAATTTATTATAC-3’.

The 2 PCR products were purified and co-transformed into yeast and selected on G418 plates. A correct insertion was screened by junction PCR using the primer pair 5’CTGGTAGAGGACAAGGTG and 5’AATGCATGCTTATAATGAGTATG. Positive clones were subjected to the cassette recycling procedure as described above. A precise removal of the cassette was verified by junction PCR using the primer pair 5’AGAAGCTAGAAAACGTGAATAC and 5’TTTAATCATATATATCTCCTTTGG. Multiple positive clones were isolated and subjected to transplantation. Genome Transplantation Protocol We list the complete genome transplantation protocol used in this paper. There are a number of modifications from the method we originally reported in 2007 (8) and 2009 (37). This protocol includes an explanation of how we generated a Mycoplasma capricolum subspecies capricolum strain California kid mutant with no restriction enzyme activity (Mcap RE(-)). This is the recipient cell strain used in all genome transplantation experiments reported here. Transplantation of M. mycoides genomic DNA cloned in yeast as YCps from melted agarose plugs into Mcap RE(-) recipient cells using the 5% polyethylene glycol (PEG)-mediated protocol Mycoplasma mycoides genomic donor DNA transplantation into Mcap RE(-) recipient cells was performed as we have previously described with a few modifications, allowing an improvement of the transplantation efficiency (8, 37). (i) Preparation of Mcap RE(-) recipient cells. We use 9 ml of Mcap RE(-) cells grown in SOB(+) at 30 °C until the pH of the culture reaches pH 6.5 (~ 5x 107 cells/ml). SOB (+) medium is Bacto SOB medium (BD Cat # 244310) supplemented with fetal bovine serum to 17%, glucose to a concentration of 10 g/l, phenol Red to 0.002%, and Penicillin G to 0.5 µg/ml. The cells are centrifuged at 4575g for 15 minutes at 10 °C, washed once in 6 ml of S/T buffer (10mM Tris-HCl pH 6.5; 250mM NaCl), then resuspended in 400 µl of 0.1 M CaCl2, and incubated on ice for 30 minutes before use or storage. These cells can be stored 4 °C for two weeks without losing transplantation competence. Alternatively, they can be frozen at -80 °C indefinitely if they are first washed with Tris-sucrose buffer (10 mM Tris pH 7.4 + 272 mM sucrose) or Hepes –sucrose buffer (8 mM Hepes pH 7.4 + 272 mM sucrose) and then stored in either of those buffers plus 10% glycerol.

   

30    

(ii) Donor genomic DNA is prepared in agarose plugs. Yeast strains harboring donor genomes as yeast centromeric plasmids (YCpMmyc) DNA are grown at 30 ºC in selective medium until the OD600 reached ~ 1.5. Cultured yeast cells with M. mycoides YCpMmyc DNA are prepared using the BioRad CHEF Mammalian Genomic DNA Plug Kit, following the instructions recommended by the manufacturer for yeast DNA isolation, with modifications as noted below. We use 6x109 yeast cells per ml of plugs, instead of 6x108 cells as recommended by the kit, in order to increase the amount of M. mycoides YCpMmyc DNA in each plug. Also, we replace the lyticase provided in the BioRad kit with Zymolyase 100T from USB Biological. We add the Zymolase enzyme agarose mixture to make the plugs and to the lyticase buffer provided with the kit at 5 mg/ml. Plugs are incubated for 2 hrs at 37 °C. After a wash in 1X TE buffer [2mM Tris-HCl pH 8.0; 5mM EDTA], embedded yeast cells are lysed, and proteins digested by two incubations of 24 hr each at 50 °C with 5 ml of Proteinase K Reaction Buffer [100 mM EDTA; 0.2% sodium deoxycholate; 1% sodium lauryl sarcosine; pH 8.0] supplemented with 200 µl of Proteinase K, per ml of plug.

The agarose plugs are washed at room temperature 4 times for 1 hr each with 40 ml of 1X TE buffer with agitation and stored in the same buffer at 4 °C. When yeast plugs are to be digested with restriction enzymes to degrade the yeast chromosomes, we add phenylmethanesulfonylfluoride (PMSF) to 1mM during the second wash. When donor genomic DNA is isolated from Mycoplasma mycoides (tetM, lacZ) bacterial cells instead of from yeast cells, the mycoplasma cells are grown in SP4 medium, supplemented with 10 µg/ml of tetracycline and 10 µg/ml of streptomycin at 37 °C until the pH of the medium reaches 6.5 (~109 cells/ml). Prior to collection of the cells, we add 50 µg/ml of chloramphenicol to the medium. In Escherichia coli, chloramphenicol is used to synchronize ongoing rounds of chromosomal replication and inhibit further rounds of replication (51-54). Chloramphenicol is also known to compact nucleoids (55, 56). The presence of chloramphenicol in mycoplasma cultures may help to increase the fraction of compact and fully replicated genomes in the agarose plugs, and has improved our genome transplantation efficiency. Immediately before the transplantation reaction, the agarose plugs containing genomic DNA are washed 4 times for 30 minutes each in 0.1X TE buffer under gentle agitation. The buffer is then completely removed and the agarose plugs are melted with 1/10 volume of 10X β-Agarase Buffer [10 mM Bis Tris-HCl pH6.5; 1 mM Na2EDTA]. There are two initial melting steps: 42 °C for 10 minutes, followed by 65 °C for 10 minutes. Then molten agarose is cooled to 42 °C for 10 minutes and incubated overnight at this temperature with 3 units of β-Agarase I (NEB) per 100 µl of plug. The quality of the genomic DNA in a plug is important, but the quantity of genomic DNA present in an agarose plug is also crucial. Using our reaction conditions, M. mycoides genomic DNA agarose plugs at a concentration of ~100 ng DNA/µl are efficient in transplantation. (iii) Transplantation reaction. After recipient cells have incubated at least 30 minutes on ice, 200 µl of cells are gently mixed with 800 µl of SP4 (-) [0% fetal bovine serum, 0.45% NaCl], and then 100 µl of melted donor genomic DNA agarose plugs is added. The transplantation reaction mixture is then incubated for 90 minutes at 37 ºC. An equal volume (1300 µl) of 2X fusion buffer

   

31    

[20 mM Tris-HCl pH 6.5; 250mM NaCl; 20mM MgCl2; 10% PEG 6000 (Fluka)] is then immediately added to the mixture of SP4(-), genomic DNA, and recipient cells. The reaction is then homogenized by rocking the tube gently four times. Then the cells are centrifuged at 4575g for 15 minutes at 10 °C, resuspended in 1.2 ml of SP4, and plated on two 10 cm plates each containing 10 ml SP4 agar medium containing 4 µg/ml of tetracycline and 150 µg/ml of X-gal. After 3-4 days of incubation at 37 ºC, individual colonies are picked and grown in broth medium containing 10 µg/ml of tetracycline. We currently obtain between 500 and 1500 transplants per transplantation reaction. Lower efficiencies are obtained using plugs from yeast cells. (iv) Cloning procedures and generation of the Mcap RE(-) strain. This was done as described in Lartigue, et al. 2009 (37). Some vectors used in this study were derived from an oriC plasmid (57) that is capable of replicating in M. mycoides (pMYCO1) or in M. capricolum (pMYCO1 and pSD4). These plasmids are based on the pBS(+) plasmid (Stratagene) and contain the tetM gene from transposon Tn916 driven by the spiralin promoter, as a resistance marker (58). The heterologous pSD4 Spiroplasma citri oriC plasmid was previously shown to transform M. capricolum. This plasmid is efficient for integration of foreign sequences or for targeted mutagenesis in the M. capricolum genome. To inactivate the potential restriction enzyme gene (MCAP0050) in M. capricolum, an internal fragment of this gene was amplified from M. capricolum genomic DNA using oligonucleotides RE-Mcap-F (5′- GATCTCTAGACTAATGTTCAATTGGATGATATAG-3′) and RE-Mcap-R (5′- GATCTCTAGACTCAAGTCTTGTAGGAGAATC-3′) that include an XbaI site (underlined). After cleavage by XbaI, the amplification product was cloned into XbaI cleaved pSD4 plasmid (59), yielding pSD4-ΔMcap0050. Ten micrograms of this plasmid was used to transform wild type M. capricolum cells using the 5% PEG mediated protocol (described below). Transformants were selected on SP4 agar plates containing 4 µg/ml tetracycline. After 7 days of incubation at 37 ºC, several colonies were grown in liquid medium containing 10 µg/ml tetracycline and cells were propagated for 15 passages. Then, clones were analyzed by Southern blot to verify the presence of the plasmid and its possible integration at the target gene by homologous recombination via a single crossover event. M. capricolum RE(-) clone 10 15P was selected because its hybridization pattern demonstrated the interruption of the MCAP0050 gene by the plasmid. No free plasmid was detected. Using centrifuge-clarified lysate extracts, we also observed a complete absence of restriction enzyme activity in that clone compared to wild type M. capricolum. During this study, M. capricolum RE(-) clone 10 was used as a source of crude cellular extracts for in vitro methylation experiments. It was not possible to use M. capricolum RE(-) clone 10 as a potential recipient cell for transplantation experiments because it contained the same resistance marker gene (tetM) as our M. mycoides donor genome. Therefore, using the same strategy, we constructed another M. capricolum RE(-) mutant carrying the YCp and puromycin gene instead of the tetM gene. M. capricolum RE(-) clone 17.5 15P was selected as the recipient for transplantation experiments because it contained no restriction enzyme activity. Comments on our current genome transplantation protocol. Here are the significant alterations of the original genome transplantation protocols

   

32    

published in 2007 and 2009 (8, 37). • Recipient cell growth temperature: Growing the Mcap RE(-) recipient cells at

30°C instead of 37°C greatly improves their competence for genome transplantation.

• Polyethylene glycol: We tested the importance of the PEG during the transplantation reaction by changing its molecular weight, concentration, and manufacturer in the 2X fusion buffer. Our results showed that the use of PEG-6000 from Fluka instead of PEG-8000 from USB clearly increased the efficiency of transplantation.

• Incubation of transplantation reaction: We also found that changing the incubation time from 50 minutes to 90 minutes at 30°C improved transplantation efficiency.

• Elimination of 3-hour recovery time at 37 °C after the transplantation reaction before plating the cells: This step was of no benefit and was eliminated from the procedure.

   

33    

Figures S1 to S21

5’AGATGTGTATAAGAGACAG 19 bp 5’CTGTCTCTTATACACATCT 19 bp 5’GAGCCAATATGCGAGAACACCCGAGAA SqRP 5’GCCAACGACTACGCACTAGCCAAC SqFp 5’TTTAATAATAAAAAATCGGGATTTCCCGATTTTTTTG Bidirectional terminator (ter) 5’CAAAAAAATCGGGAAATCCCGATTTTTTATTATTAAA Bidirectional terminator (ter) CTGTCTCTTATACACATCTCAACCATCATCGATGAATTTTCTCGGGTGTTCTCGCATATTGGCTCGAATTCGAGCTCGGTACCC|TTTAATAATAAAAAATCGGGATTTCCCGATTTTTTTGTATTTTTTGAATTAAGTATTAAATAAGTGTAAAATATATAATAGTAAAAACGCCCCAAAAGGGGCAGACAAAATAGTAGAAATATATCTATTATTCTTGATTTGTTATAGAAATTTTAAGGAGAAAAAAACatgactgaatataaacctactgttagattagctactagagatgatgttcctagagctgttagaactttagctgctgcttttgctgattatcctgctactagacatactgttgatcctgatagacatattgaaagagttactgaattacaagaattatttttaactagagttggtttagatattggtaaagtttgggttgctgatgatggtgctgctgttgctgtttggactactcctgaaagtgttgaagctggtgctgtttttgctgaaattggtcctagaatggctgaattaagtggtagtagattagctgctcaacaacaaatggaaggtttattagctcctcatagacctaaagaacctgcttggtttttagctactgttggtgttagtcctgatcatcaaggtaaaggtttaggtagtgctgttgttttacctggtgttgaagctgctgaaagagctggtgttcctgcttttttagaaactagtgctcctagaaatttacctttttatgaaagattaggttttactgttactgctgatgttgaagttcctgaaggtcctagaacttggtgtatgactagaaaacctggtgcttaaCAAAAAAATCGGGAAATCCCGATTTTTTATTATTAAA|GGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCCAACGACTACGCACTAGCCAACAAGAGCTTCAGGGTTGAGATGTGTATAAGAGACAG Fig. S1 Structure and sequence of transposon Tn5 puro containing the puromycin resistance gene. DNA with the sequence internal to the two vertical marks “|” was ligated into the SmaI site of the multiple cloning site (MCS) in the EZ-Tn5 pMOD2 construction vector (Epicentre) to form the complete transposon sequence. EZ-Tn5 pMOD2 containing the transposon was digested with PshAI (New England Biolabs) and gel purified to obtain the Tn5 puro DNA. Ptuf is the tuf promoter. Ter red triangle is a bidirectional terminator that is downstream of the gyrA gene of syn1.0. SqPR and SqPF green triangles are primer sites for inverse PCR. 19 bp blue triangles are the Tn5 inverse termini to which Tn5 transposase binds to form the activated transposome used for generating Tn5 mutagenesis libraries.

   

34    

Step 1. Construct Tn5 transposon containing 19 bp mosaic ends, sequencing

primer sites, terminator sequences, and a selectable marker (puromycin-resistance gene). Bind Tn5 transposase (Epicentre) to the 19bp termini to form the active transposome.

Step 2. Introduce transposome into Mycoplasma mycoides JCVI-syn1.0 R-M

(minus) strain by polyethylene glycol (PEG) transformation method. Collect puromycin-resistant colonies and serially propagate to eliminate slow growers.

Step 3. Isolate P0 and P4 genomic DNA, shear using a nebulizer, ligate to circularize fragments. PCR amplify specific fragments (inverse PCR) and sequence these fragments using MiSeq.

Fig. S2 Steps in producing a Tn5 global insert library.

~80,000

colonies

       

     

                               

       

   

           

               

                           

             

 

~103                                

~104                              

~104                                

~104

Prepare  Library  P0  from  DNA  isolated  from  colonies.    All  viable  insertions  represented.

Prepare Library P4 from final passage (~50 doublings). Slow growers are lost.

   

35    

Fig S3. Genes can be placed into 3 categories. Genes that were hit frequently by both P0 and P4 insertions were classified as non-essential n-genes. Genes hit primarily by P0 insertions but not P4 insertions were classed as quasi-essential, growth impaired i-genes. Genes that were not hit at all, or were sparsely hit in the terminal 20% of the 3’-end or the first few bases of the 5’-end were classed as essential e-genes. The first complete gene in the figure is a quasi-essential i-gene. The second gene is an essential e-gene. The third complete gene at bottom of figure is classed as an n-gene. Library P0, black bars; library P4, red bars.

   

36    

Fig. S4 Syn1.0 gene map with Tn5 P4 insertions showing clustering of non-essential genes. Protein-coding genes are indicated as blue arrows and RNA-coding genes as red arrows. Fine black marks indicate P4 Tn5 insertions. P4 insertions most clearly identified the n-genes since e-genes and i-genes had no hits or were sparsely hit, respectively (Fig S3A). Non-essential genes tended to occur in clusters (yellow arrows) far more than expected by chance. The yellow arrows indicate the deletions in the RGD1.0 design. Regions of the map between the yellow arrows are mainly occupied by e-genes and i-genes.

   

37    

Fig. S5. The M. mycoides JCVI-syn1.0 genome (1078kb) displayed using CLC software and showing 26 genes added back to yield RGD2.0. Dark blue arrows are protein coding genes and red arrows are RNA genes. Brown arrows are the 8 segments. Purple arrows are the regions kept in the RGD2.0 design and yellow arrows are deleted regions. Green arrows indicate regions added back to the RGD1.0 design to produce the RGD2.0 design.

   

38    

Fig. S6 List of genes deleted in the RGD2.0 design of segment 5. To arrive at the final structure of the seg 5 used in JCVI-syn2.0, we started with syn1.0 seg 5 and carried out scarless (TREC) deletions of cluster 33, 36, and 37. We then replaced the 2 genes 0487 and 0488 that were between cluster 36 and 37 with gene 0154, which had been deleted from segment 2, but had converted to strong “i” in the RGD2678 intermediate assembly. These changes to segment 5 resulted in the viable cell, syn2.0.

   

39    

Fig. S7 Three design cycles involved in building syn3.0. The map shows details of the deleted and added back genes in the various cycles. The starting cell is syn1.0 (1,078,809 bp). Dark blue arrows indicate the syn1.0 genes. Brown arrows indicate the 8 segments with 200 bp overlaps. Design cycle 1: Yellow arrows indicate deletions in the RGD1.0 design. RGD1.0 was not viable, but a combination syn1.0 segments 1,3,4,5 and RGD1.0 segments 2,6,7,8 was viable (referred to as RGD2678 in text). Design cycle 2A: The green arrows indicate 26 genes that were added back to RGD1.0 segments 1,3,4,5 in an attempt to enable those segments to give a viable cell in combination with RGD2678. This version was only viable when syn1.0 segment 5 was substituted for the RGD1.0 add back version. Design cycle 2B: As described in the text, deletions of genes 454-474 and 483-492 from the syn1.0 segment 5 yielded a viable RGD2.0 (576,028 bp). This was equivalent to adding back additional genes (blue arrows) to RGD1.0 segment 5 and deleting genes 487-488 (tan arrow). Not shown is the insertion of gene154 in place of

   

40    

487-488. Design cycle 3: The red arrows indicate an additional 37 syn1.0 mycoplasma genes plus 2 vector genes (bla and lacZ) and the rRNA operon in segment 6 that were deleted from syn2.0 to produce a viable syn3.0 cell (531,560 bp).

Fig. S8 Diagram of the TREC deletion method (38). To generate a scarless deletion, the CORE cassette was PCR-amplified in two rounds to produce the knock-out cassette contains a 50 bp (“U” block) for homologous recombination, 50 bp (“D” block) repeated sequence, and a 50 bp (“D” block) for homologous recombination. (1) The cassette was transformed into a yeast strain harboring a mycoides genome and selected on SC- plate. Correct target knock-out was identified by PCR screening for insertion junctions (L and R). (2) Galactose induction results in the expression of I-Sce I endonuclease, which cleaves the 18-bp I-Sce I site (red bar) to create a double-strand break that promotes homologous recombination between two tandem repeat sequences (“D” block). (3) Recombination between two repeat sequences generates a scarless deletion. The deletion of a target region was confirmed by PCR using primers located up and down stream of the target region.

   

41    

A  

B  

   

42    

Fig. S9 Diagram of method for creating eight NotI strains. (A) The syn1.0∆RE∆IS genome was used to place pairs of NotI sites around the genome to create 8 different NotI strains. The genome in each strain was engineered by the TREC method (38) in yeast with only two unique NotI restriction sites flanking an approximately 1/8th segment of the genome. For instance, two NotI sites in the genome of NotI strain 1 were generated at locations of NotI -1U and NotI -1D, indicated by red arrows, and in the genome of the NotI strain 2, locations of NotI site were generated at the of NotI-2U and NotI -2D, etc. Each of the 1/8th segment overlaps adjacent segments by 200 bp for genome assembly. A non-essential region (24,916-bp, from gene MMSYN1_0889 to 0904), flanked by two NotI sites (NotI -8D and NotI -1U), was not included in the design (indicated in light blue). Thus, a 200 bp overlapping region, represented by vertical line, was inserted upstream of the NotI site, located at the NotI -8D. One non-essential region (from MMSYN1_0550 to 0591), indicated in gray, was replaced with the yeast selection marker MET14 in the genome of the NotI strain 6. Eight NotI -engineered genomes were transplanted from yeast to M. capricolum recipient cells to produce 8 NotI mycoides strains. (B) 1/8th genome segments could be released from mycoides genomes by restriction enzyme NotI and assembled in yeast. (C) 8 NotI -digested genomic DNA (from 1 to 8) were subjected to 1% agarose gel electrophoresis to separate 7/8th and 1/8th genome (top). The 1/8th genome segments were recovered by electro-elution from the agarose gel and analyzed in 1% agarose gel electrophoresis (bottom).

   

43    

Fig. S10 Construction of the 1/8th RGD +7/8 th syn1.0 genome by Recombinase-mediated cassette exchange (RMCE, (18). 1) An approximate 1/8th genome (colored with gray) of the M. mycoides JCVI-syn1.0 cloned in yeast was replaced with a landing pad cassette flanked by two 34-bp hetero-specific lox sites. (2) A donor plasmid containing a corresponding 1/8th RGD, flanked by another two hetero-specific lox sites was introduced into to the landing pad strain by yeast mating. 3) The 1/8th RGD was exchanged with the landing pad locus via Cre-mediated recombination. An intron-containing the URA3 gene was split between the landing pad and the donor plasmid so that the cassette exchange could be selected by restoration of uracil prototrophy. 4) The 1/8th RGD+ 7/8th syn1.0 genome was transplanted into M. Capricolum recipient cells to produce a mycoides strain. 5) A 1/8th RGD fragment can be purified by NotI digestion and then used for genome assembly (Material and methods).

   3’  URA3

 MET14

     

 

  1/8th  RGD      

 

 5’  URA3

TRP1

 Cre

 P

Donor  vector

 

 

 5’  URA3

TRP1

 Cre

 P

 3’  URA3

 MET14

     

2

3

+

 

 

1/8th  

RGD  

 

 

 3’  URA3

 MET14

     

1

WT  Syn1.0

     hetero-­‐specific  lox  sites

4

   

 

1/8th  

RGD   5

   

44    

Fig. S11 Schematic diagram of genome engineering to produce syn2.0 in yeast. Deletion of gene clusters 33, 36 and 37 (See Table S10) in segment 5 of the 7RGD+ syn1.0-5 genome was produced by 2 rounds of deletions using the CORE6 cassette containing KlURA3 and TRP1 markers, respectively as follows: 1) The CORE6 cassette was seamlessly recycled via homologous recombination between 2 repeated sequences flanking the cassette. 2) The CORE6 cassette was then used again to replace the region covering the TRP1 marker, genes MMSYN1_0487 and_0488, and cluster 37. 3) MMSYN1 154 was inserted into the genome via a knock-in module followed by cassette recycling (50).

   

45    

Fig. S12 Gel analysis of twenty-four representative 1.4 kb dsDNA fragments assembled during the construction of HMG. Panel A shows the dsDNA fragments following oligonucleotide assembly and amplification and Panel B shows the same dsDNA fragments following error correction and PCR amplification. Two microliters of each sample were loaded onto a 1% E-gel and run for 20 minutes. M indicates the 1kb ladder (NEB).

   

46    

Fig S13. Clone collapsing for scalable cassette identification. Twenty-four bacterial clones for each cassette were grown separately in liquid culture in 96-well plates and then collapsed into a single set of 24 wells. Each color grouping represents a single cassette while the green grouping is the final collapsed group.

   

47    

Fig S14. Rolling Circle Amplification (RCA) products derived from the HMG eighth molecule assemblies. Supercoil DNA was extracted from yeast clones containing the HMG eighth molecule assemblies and used as template in RCA reactions with either the (A) GE-Templiphi Large Construct kit or (B) Qiagen-REPLI-g kit. The RCA products were digested with NotI and separated on an agarose gel subjected to field-inverted gel electrophoresis (FIGE) using the U-9 program as previously described (7). The expected insert size for each eighth molecule is indicated above each lane.

   

48    

Fig S15. Field-inverted gel electrophoresis analysis of HMG. Yeast clones harboring HMG (lane 2) were purified from yeast in agarose plugs as previously described (Gibson et, 2008), digested with AscI to linearize the 483-kb genome, and then analyzed by FIGE using the U-2 program (7). The same analysis was performed with yeast not harboring HMG as a negative control (lane 1). M indicates the lambda ladder (NEB).

   

49    

Fig S16. Schematic for editing previously generated sequence-verified cassettes. Cassettes (Blue rectangles) generated during the construction of HMG, RGD1.0, and RGD2.0 were manipulated to remove genes (red square), add genes (green square), and make single nucleotide substitutions (orange circle). This was readily performed by generating overlapping fragments via PCR (blue arrows) and then assembling the resulting fragments in vitro.

   

50    

Fig S17. Correlation of PicoGreen fluorescence with cell concentration. Relative fluorescence unit (RFU) measurements were obtained from a late logarithmic phase culture of JCVI-syn1.0 cells diluted with SP4 medium in a 2-fold series (right to left) prior to processing as described in materials and methods. Medium controls generated a value, RFU=14, that was subtracted from each sample to give the net RFU values shown.

R²  =  0.9999  

40  

80  

160  

320  

640  

1280  

2560  

2   3   4   5   6   7   8   9  

RFU  

   

51    

Fig S18. RGD1.0, Segment 6 (+ 7/8 syn1.0) grows slowly and produces sectored colonies after 10 days. Colonies from transplantation of three different yeast clones of RGD1.0 Segment 6 + 7/8 syn1.0 are shown. Generally, colonies could be seen in 3 to 4 days after genome transplantation. The one exception, segment 6, initially was detected as a very small colony which after 10 days produced faster growing sectors. The sequence of several independent clonal isolates from the sectored colonies revealed mutations that reduced the stability of a stem-loop transcription terminator downstream of the tRNA-His gene. This allowed read through and expression of the essential gene 0621. Figure S19 shows the sequence of this region in syn1.0 and as designed in RGD1.0 segment 6. Figure S20 and Figure S21 show the various mutations that restored expression of gene 0621 and yielded a viable segment 6.

   

52    

Fig S19. Mutations in Segment 6 sequence reduce the stability of a stem-loop transcription terminator downstream of the tRNA-His gene. At the top the relevant sequence region is shown for syn1.0 and at the bottom is the segment 6 design sequence. The sequence of several independent clonal isolates (shown in Fig S18) revealed mutations that reduced the stability of a stem-loop transcription terminator downstream of the tRNA-His gene. This allowed read through and expression of the essential gene 0621. Fig S20 and Fig S21 show the various mutations that restored expression of gene 0621 and yielded a viable segment 6.

   

53    

Fig S20. Mutations in Segment 6 +7/8 in colony purified isolates from transplant of clone #5 (shown in Fig S18) .

   

54    

Fig S21. Mutations of tRNA-His terminator in RGD 1.0 Seg6+7/8 transplants (mutations at GC base pairs in the stem; see also Fig S18-S20).

   

55    

Tables S1 to S15

Table S1. RGD2.0 design compared to RGD1.0. Sizes and fractional sizes of the 8 segments are listed for the RGD1.0 design and the RGD2.0 design. The final genome lengths are corrected for the 200 bp overlaps between the segments. Segments 1, 3, 4, and 5 are redesigned in RGD2.0, whereas segments 2, 6, 7, and 8 remain the same for both designs. Genes deleted in the RGD1.0 design are indicated by yellow arrows in Fig S5. The 26 genes added back to RGD1.0 to yield the RGD2.0 design are listed in Table S2 and shown in Fig S5.

   

56    

Table S2. 26 genes were identified for add back to RGD1.0 segments 1, 3, 4, and 5 to yield the new RGD2.0 design (See Table S1). We used two methods to identify genes for add back: (1) Tn5 mutagenesis of RGD2678. The Tn5 mutagenesis data for RGD2678 is shown for lib P0 (0g) in column 7 and lib P4 (4g) in columns 8. Columns 3 and 4 show Tn5 mutagenesis data for syn1.0 and columns 3 and 4 show data for D5 (Table S6). (2) Analysis of the viability of 39 cluster deletions (Table S8) in clone 19 and clone 59 (Table S7). Genes that produced non-viable deletions (nv) are in columns 9 and 10 (pink highlight).

   

57    

Table S3. 37 non-essential genes selected for deletion from syn2.0 to yield syn3.0 Tn5 mutagenized syn2.0 cells were passaged 6 times to deplete quasi-essential genes. The number of Tn5 insertions in each gene are shown for passages P0, P1, P2, and P6. Persistance of high numbers of Tn5 inertions during the passages is characteristic of non-essential genes.

MMSYN1 SGI  Annotation syn2_P0 syn2_P1 syn2_P2 syn2_P6  _0013  Mycoides  cluster  lipoprotein,  LppA/P72  family 168 88 145 93  _0028  Cold-­‐shock  DNA-­‐binding  protein  family 17 12 12 6  _0031  Heat  shock  protein  33,  redox  regulated  chaperonin 31 13 30 13  _0035  Variable  surface  protein 115 45 91 48  _0036  D-­‐isomer  specific  2-­‐hydroxyacid  dehydrogenase,  NAD  binding  domain98 52 73 62  _0037  Transporter,  auxin  efflux  carrier  (AEC)  family  protein 97 59 77 62  _0038  ATPase  (AAA+  superfamily) 106 58 69 60  _0048  Cytidine  and  deoxycytidylate  deaminase  zinc-­‐binding  region  family  protein32 16 23 13  _0062  Macrophage  Migration  Inhibitory  Factor 19 7 21 5  _0078  Alpha/beta  hydrolase  fold  family  protein 56 28 46 22  _0096  Proline  dipeptidase 17 7 6 7  _0217  Glycerol  uptake  facilitator  protein. 113 57 88 58  _0219  FAD/NAD(P)-­‐binding  domain 113 53 60 25  _0258  NAD(P)-­‐binding  Rossmann-­‐fold  domains 54 21 40 25  _0278  PTS  system  fructose-­‐specific  enzyme  iiabc  component 218 108 183 133  _0279  Membrane  protein 33 20 25 20  _0284  Lysophospholipase  Monoglyceride  lipase 30 18 19 15  _0333  Lipoprotein,  putative  (VlcA) 24 6 28 18  _0334  Lipoprotein,  putative  (VlcB) 34 13 34 25  _0335  Lipoprotein,  putative  (VlcC) 37 13 30 17  _0336  Phosphotransferase  system  PTS,  IIA  component 26 10 16 11  _0351  Holliday  junction  resolvase  RecU 13 2 11 6  _0355  Lipoprotein,  PARCEL  family 42 16 36 31  _0370  Single-­‐strand  binding  family  protein 23 14 18 12  _0417  Prophage  protein  (Ps3) 23 13 24 38  _0436  Uracil-­‐DNA  Glycosylase|  subunit  E 34 14 24 16  _0446  Membrane  protein 19 6 6 9  _0476  N-­‐acetylglucosamine-­‐6-­‐phosphate  deacetylase 75 44 57 27  _0477  Membrane  protein 27 20 23 13  _0480  Conserved  predicted  protein 90 71 100 135  _0496   8 3 9 4  _0497  Solute:sodium  symporter  (SSS)  family  transporter 117 57 98 74  _0498  N-­‐acetylneuraminate  lyase 70 35 46 30  _0514  Membrane  family  protein 46 25 24 24  _0677  Membrane  protein 38 27 30 26  _0829  Hydrolase,  TatD  deoxyribonuclease  family  protein 47 18 31 18  _0905   6 2 10 4

   

58    

Genome Construction # Full-length RGD3 constructs (out of 48)

Transplantation Results

(E) RGD3 𝝙 rDNA operon I 3 0 out of 3 (F) RGD3 3 3 out of 3 (G) RGD3 𝝙 rDNA operon II 10 2 out of 10

Table S4: RGD3 genome constructions in yeast and transplantation results. Eighth molecule RGD3-1 was synthesized with and without rDNA operon I and eighth molecule RGD3-6 was synthesized with and without rDNA operon II in order to generate three RGD3 genomes: (E) absence of rDNA operon I, (F) presence of rDNA operons I and II, and (G) absence of rDNA operon II. Although full-length RGD3 genomes were assembled in yeast for all three, only (F) and (G) genome versions could generate transplants. One transplant from (G), assigned clone 19-1, was further characterized and later named syn3.0.

   

59    

A

g19P

0_F

g19P

0_R

g19P

1_F

g19P

1_R

g19P

4_F

g19P

4_R

P4_F+P

4_R

MMSYN1

HSW  13

0821

Hand

 anno

tation

8 8 5 8 1 1 2  _0400 in ThiJ/PfpI  family  protein3 1 10 3 2 1 3  _0004 in rsmA,  ksgA,  16S  rRNA  m6  2A1518,  m6  2A15194 1 3 1 2 1 3  _0376 in conserved  hypothetical  protein2 2 7 10 1 2 3  _0401 in peptidase  C39  family  protein4 3 5 3 1 2 3  _0504 in rsmI,  16S  rRNA  Cm14021 1 6 6 2 2 4  _0409 in NIF3  family  protein1 2 2 5 2 2 4  _0851 in real?5 3 12 8 3 2 5  _0416 in conserved  hypothetical  protein1 1 5 2 2 3 5  _0113 in glycosyltransferase0 3 0 11 1 4 5  _0421 in conserved  hypothetical  protein6 5 11 7 4 2 6  _0381 in MTA/SAH  nucleosidase4 5 1 8 1 5 6  _0046 in recR6 2 8 6 5 2 7  _0382 in deoxynucleoside  kinase2 4 14 10 5 3 8  _0326 in conserved  hypothetical  protein3 4 11 9 3 5 8  _0495 in ROK  family  protein5 3 3 1 6 3 9  _0214 in PAP2  superfamily  domain  membrane  protein6 1 6 6 5 4 9  _0114 in glycosyltransferase3 0 6 6 5 4 9  _0852 in real?7 7 11 11 4 5 9  _0838 in rlmB,  23S  rRNA  Gm225136 25 18 15 4 6 10  _0095 in secA14 19 27 26 7 4 11  _0127 in HD  domain  protein19 16 22 21 6 5 11  _0264 in serine/threonine  protein  kinase6 8 13 19 5 6 11  _0517 in rluD13 18 12 23 3 11 14  _0042 in transcriptional  regulator,  RpiR  family  protein6 8 17 14 5 10 15  _0907 in conserved  hypothetical  protein3 9 5 15 2 13 15  _0080 in conserved  hypothetical  protein9 7 18 14 12 6 18  _0043 in might  be  rsmC  or  rlmF15 5 16 9 11 8 19  _0005 in real?9 10 20 22 11 8 19  _0494 in putativeN-­‐acetylmannosamine-­‐6-­‐phosphate2-­‐epimeras22 28 40 45 9 13 22  _0305 in Xaa-­‐Pro  peptidase,  M24  family67 72 61 73 10 15 25  _0824 in uvrA12 16 19 31 11 17 28  _0732 in deoxyribose-­‐phosphate  aldolase55 53 69 55 18 16 34  _0825 in uvrB

   

60    

B

52 46 45 23 0 2 2  _0316 i transketolase7 4 0 0 0 2 2  _0394 i ATP-­‐dependent  protease  La4 3 0 5 0 2 2  _0525 i protein  MraZ30 31 11 17 0 2 2  _0799 i glycine  hydroxymethyltransferase10 11 14 14 0 2 2  _0823 i folC  folate  synthetase-­‐polyglutamyl  folate  synthetase24 21 16 13 0 2 2  _0872 i ychF17 9 10 10 1 2 3  _0240 i thiI,  s4U  modification  in  tRNA  with  icsS1 3 6 4 0 3 3  _0777 i conserved  hypothetical  protein12 24 23 14 2 2 4  _0887 i cdr6 15 4 13 1 3 4  _0132 i AAA  family  ATPase3 5 12 16 1 3 4  _0239 i conserved  hypothetical  protein1 0 8 4 4 2 6  _0814 i UDP-­‐galactopyranose  mutase27 20 25 17 3 3 6  _0414 i RelA/SpoT  family  protein3 7 5 10 3 3 6  _0620 i ferric  uptake  regulator6 6 23 29 3 5 8  _0479 i conserved  hypothetical  protein2 6 3 10 3 5 8  _0817 i conserved  hypothetical  protein  (WhiA)8 4 14 9 7 3 10  _0142 i protein-­‐(glutamine-­‐N5)methyl  transferase,  release  factor7 3 21 11 7 4 11  _0347 i cytidylate  kinase10 20 6 24 1 10 11  _0108 i putative  lipoprotein3 4 15 14 7 5 12  _0404 i recO2 5 6 12 5 7 12  _0853 i conserved  hypothetical  protein15 14 20 17 8 5 13  _0697 i glycosyltransferase48 40 43 46 6 10 16  _0708 i alkyl  phosphonate  ABC  transporter,substrate-­‐bindin22 29 44 41 6 10 16  _0878 i amino  acid  permease8 13 23 22 15 5 20  _0329 i rluB  n23  or  rsuA  n2321 15 27 31 14 11 25  _0434 i tRNA:M(5)U-­‐54  methyltransferase16 18 25 30 2 23 25  _0106 i xseA13 14 20 29 11 17 28  _0435 i phosphomannose  isomerase  type  I9 15 26 27 10 18 28  _0216 i hypoxanthine  phosphoribosyltransferase13 13 26 26 17 13 30  _0097 i dna  polymerase  I,  5'-­‐3'  exonuclease29 17 40 35 25 17 42  _0876 i amino  acid  permease3 2 13 16 23 30 53  _0115 i utp–glucose-­‐1-­‐phosphateuridylyl  transferase(udp-­‐g80 77 83 96 33 28 61  _0228 i pdhD20 27 40 42 31 31 62  _0433 i copper  homeostasis  protein46 43 69 70 30 39 69  _0133 i conserved  hypothetical  protein14 10 47 42 42 29 71  _0411 i putative  membrane  protein89 76 94 91 46 35 81  _0227 i pdhC9 2 21 14 53 41 94  _0733 i phosphoglucomutase  or  phosphomannomutase

   

61    

C

D

Table S5. Tn5 mutagenesis of syn3.0 with genes listed that show significant numbers of inserts after four serial passages (P4). The genes are sorted into 4 groups A, B, C, and D based on their original syn1.0 classifications as in, i, ie, or n, respectively. They are further sorted according to numbers of P4 inserts (from small to large). Columns 1 and 2 show P0 forward and reverse orientation inserts, columns 3 and 4 are for passage P1, and columns 5 and 6 are for passage P4. Column 7 shows the sum of forward and reverse oriented insertions for P4. Column 8 is the gene name, column 9 is the original syn1.0 gene classification based on Tn5 mutagenesis, and column 10 is the gene functional annotation.

0 1 3 2 1 1 2  _0789 ie ATP  synthase,  delta  subunit1 1 1 1 0 2 2  _0067 ie 5S  ribosomal  RNA8 20 6 7 0 2 2  _0068 ie 23S  ribosomal  RNA0 1 1 3 0 2 2  _0910 ie ribosomal  protein  L340 5 2 14 1 2 3  _0301 ie rimP0 3 1 6 1 2 3  _0873 ie conserved  hypothetical  protein2 2 0 2 0 3 3  _0693 ie conserved  ,  caax  amino  protease  family0 3 10 4 3 1 4  _0346 ie conserved  hypothetical  protein3 2 6 2 3 1 4  _0632 ie conserved  hypothetical  protein9 5 15 9 8 2 10  _0481 ie lipoprotein,  putative  (VlcE)2 2 12 8 6 5 11  _0726 ie glucosamine-­‐6-­‐phosphate  deaminase8 10 9 27 13 35 48  _0109 ie apurinic  endonuclease  Apn1

2 7 2 7 1 2 3  _0874 n 16S  rRNA  methyltransferase  GidB6 5 13 16 4 0 4  _0640 n tRNA  pseudouridine2 5 8 9 1 4 5  _0290 n tRNA  pseudouridine  synthase  B1 0 2 1 1 4 5  _0462 n IS1296  transposase  protein  A5 5 13 11 5 1 6  _0601 n putative  membrane  protein16 22 22 24 5 3 8  _0060 n putative  membrane  protein3 3 15 11 3 6 9  _0094 n putative  membrane  protein4 6 17 16 3 7 10  _0692 n 23S  rRNA  pseudouridine  synthase17 9 19 21 8 6 14  _0505 n putative  lipoprotein15 13 18 21 12 9 21  _0306 n hypothetical  protein12 17 31 37 9 15 24  _0444 n endopeptidase  O29 10 39 10 21 6 27  _0063 n tRNA-­‐dihydrouridine  synthase  B

   

62    

Table S6. Stepwise D-series deletions of JCVI syn 1.0. The scarless TREC (tandem repeat coupled with endonuclease cleavage) deletion method (38) was used to generate a series of reduced genomes in yeast. Six insertion elements (IS) and in addition, two genes (MMSYN1_460 and _463) flanking one of the IS elements, were sequentially deleted in the genome of JCVI-syn1.0 Δ1–6 (39) to produce the syn1.0∆RE∆IS genome. Based on Tn5 insertion data for identifying non-essential gene clusters, 22 clusters were selected and subjected to deletion sequentially in syn1.0∆RE∆IS to produce strains D1 to D22. After each round of deletion, the resulting genome was tested for viability by transplantation (8, 37).

   

63    

Table S7A 1/8 genome Note RGD segment RGD1.0-1 RGD1.0-2 RGD1.0-3

RGD1.0-3s RGD1.0-3 plus three genes (0217, 0218, and 0219)

RGD1.0-4 RGD1.0-5 RGD1.0-6 RGD1.0-6sf self-fixed isolate

RGD1.0-6sf-LP self-fixed isolate with the insertion of a landing pad

RGD1.0-6P fixed promoter

RGD1.0-6P-LP fixed promoter with the insertion of a landing pad

RGD1.0-7 RGD1.0-8 RGD2.0-1 reversion RGD2.0-3 reversion RGD2.0-4 reversion RGD2.0-5 reversion syn1.0 segment segment 1 segment 2 segment 3 segment 4 segment 5 segment 6 segment 6M The MET14 marker insertion segment 7 segment 8

   

64    

Table S7B

Assembly clone# semi-RGD colony appears (days)*

1

13 RGD27 3 91 RGD2478 4 105 RGD47 3 110 RGD46 3 124 RGD24678 4 139 RGD478 3 140 RGD2678 3 168 RGD478 3

2 11 RGD12678 6

19 RGD24*678 4 90 RGD25678 4

3 18 RGD2345678 5

32 RGD1235678 6 49 RGD1234678 4

4 48 RGD1234678 4 * Number of days after transplantation for colonies to be seen by the

naked eye. Assembly 1: (RGD1.0 1, 2, 4, 6, 7, and 8) + (syn1.0 segments 1 to 8)

Assembly 2: (RGD1.0 1 to 8) + (syn1.0-segments 1, 3, 4, 5)

Assembly 3: (RGD2.0 3, 4, and 5) + RGD1.0 2, 6, 7, and 8) + syn1.0segment 1

(RGD2.0 1, 4, and 5) + (RGD1.0 2, 6, 7, and 8) + syn1.0segment 3

(RGD2.0 1, 3, and 5) + (RGD1.0 2, 6, 7, and 8) + syn1.0segment 4

(RGD2.0 1, 3, and 4) + (RGD1.0 2, 6, 7, and 8) + syn1.0segment 5

Assembly 4: (RGD2.0 4, and 5) + RGD1.0 2, 3s, 6, 7, and 8) + syn1.0segment 1

(RGD2.0 1, 4, and 5) + (RGD1.0 2, 6, 7, and 8) + syn1.0segment 3

(RGD2.0 1 and 5) + (RGD1.0 2, 3s, 6, 7, and 8) + syn1.0segment 4

(RGD2.0 1 and 4) + (RGD1.0 2, 3s, 6, 7, and 8) + syn1.0segment 5 Table S7. Generation of intermediate RGD transplants. (A) A list of RGD and syn1.0 segments. Different versions of RGD segments were used in each assembly. (B) Various intermediate RGD transplants were isolated from 4 independent assemblies. Assemblies 1 and 2 were performed in a combinatorial manner. Assemblies 3 and 4 were carried using a combination of 7 RGD segments along with 1 syn1.0 segment. The syn1.0 segment 6M was used in Assembly 1; the RGD1.0-6sf-LP was used in Assemblies 1 and 2; and RGD1.0-6P-LP was used for Assemblies 3 and 4.

   

65    

gene or cluster segment

transplantation

colony size

1 0014-0016 Seg1 yes 2 0019-0024 Seg1 yes 3 0035-0038 Seg1 yes S 4 0041 Seg1 yes 5 0050-0060 Seg1 yes S 6 0072-0075 Seg1 yes 7 0077-0078 Seg1 no 8 0080 Seg1 yes S 9 0083-0093 Seg1 yes 10 0204-0212 Seg3 yes 11 0217-0219 Seg3 no 12 0223-0226 Seg3 yes 13 0231-0232 Seg3 yes 14 0236-0237 Seg3 yes 15 0241-0246 Seg3 yes 16 0251-0252 Seg3 yes 17 0255-0256 Seg3 yes 18 0261 Seg3 yes 19 0268-0269 Seg3 yes 20 0272-0277 Seg3 yes 21 0292-0293 Seg3 yes 22 0331-0332 Seg4 yes S 23 0349 Seg4 yes 24 0354 Seg4 yes 25 0357-0358 Seg4 yes 26 0367-0369 Seg4 yes 27 0383-0386 Seg4 yes 28 0393 Seg4 yes S 29 0395-0397 Seg4 yes 30 0417 Seg5 yes 31 0436 Seg5 yes 32 0444 Seg5 yes 33 0454-0474 Seg5 yes 34 0476-0477 Seg5 yes 35 0480 Seg5 yes 36 0483-0486 Seg5 yes 37 0489-0492 Seg5 yes 38 0494-0498 Seg5 yes 39 0503-0505 Seg5 yes S

Table  S8:  List  of  39  genes  and  cluster  deletions.  39  single  genes  and  gene  clusters  that  had  been  deleted  in  the  RGD1.0  design  were  subjected  to  individual  deletion  in  the  RGD2678  clone  140.  Deletion  of  two  clusters  (0077-­‐0078  and  0217-­‐0219)  did  not  produce  transplants  (highlighted  in  yellow).  Deletion  of  4  targets  (0080,  0331-­‐0332,  and  0393,  and  0503-­‐0505)  produced  transplants  with  a  slower  growth  phenotype  (indicated  as  “S”  in  column  5).  

   

66    

5.2" Table S9: The list of 26 genes to be added back in the redesign of RGD1.0 segments 1, 3, 4, and 5. The RGD1.0 segments 1, 3, 4, and 5 were re-synthesized with the 26 genes added back to produce RGD2.0 segments 1, 3, 4, and 5 (See also Fig S5, Tables S1, S2).

   

67    

MMSYN1_0417   cdse          MMSYN1_0436   uracil-­‐DNA  glycosylase  (UDG)          MMSYN1_0454   hypothetical  protein  MMSYN1_0455   putative  membrane  protein  MMSYN1_0924   conserved  domain  protein  MMSYN1_0460   bacterial  surface  protein26-­‐residuerepeatprotein  MMSYN1_0463   NADH  dependent  flavin  oxidoreductase  MMSYN1_0464   lipoate–protein  ligase  MMSYN1_0465   conserved  hypothetical  protein  MMSYN1_0466   glycine  cleavage  system  H  protein  MMSYN1_0467   triacylglycerol  lipase  MMSYN1_0468   lipase-­‐esterase  MMSYN1_0469   lipase-­‐esterase  MMSYN1_0470   conserved  hypothetical  protein  MMSYN1_0471   hypothetical  protein  MMSYN1_0472   putative  liporotein  MMSYN1_0473   ABC  transporter,  ATP  binding  protein  MMSYN1_0474   ABC  transporter,  ATP  binding  protein          MMSYN1_0476   N-­‐acetylglucosamine-­‐6-­‐phosphate  deacetylase          MMSYN1_0480   conserved  hypothetical  protein          MMSYN1_0483   holliday  junction  DNA  helicase  RuvA  

MMSYN1_0484  holliday  junction  ATP-­‐dependent  DNA  helicaseRuvB  

MMSYN1_0485   dihydrolipoamide  dehydrogenase  MMSYN1_0486   conserved  hypothetical  protein  MMSYN1_0487   conserved  hypothetical  protein  MMSYN1_0488   ribosome  biogenesis  GTPase  YqeH  MMSYN1_0489   DNA  polymerase  IV  MMSYN1_0490   papain  family  cysteine  protease,  putative  MMSYN1_0491   uridine  kinase  MMSYN1_0492   conserved  hypothetical  protein          

MMSYN1_0494  putativeN-­‐acetylmannosamine-­‐6-­‐phosphate2-­‐epimeras  

   

68    

Table S10. Gene clusters subjected to deletion in the 7RGD+ syn1.0 5 clone 48. See Table S7 for segment composition of clone 48. The genes are those that had been deleted in the design of RGD2.0 segment 5. The final syn2.0 genome was produced by deletion from clone 48 of clusters 33 (0454-0474), 36 (0483-0486), and 37 (0489-0492), highlighted in yellow, and replacement of genes 0487-0488 with MMSYN1_0154.

MMSYN1_0495   ROK  family  protein  MMSYN1_0496   conserved  hypothetical  protein  MMSYN1_0497   sodium:solute  symporter  family  

MMSYN1_0498  N-­‐acetylneuraminatelyase(N-­‐acetylneuraminicacidal  

       MMSYN1_0503   conserved  hypothetical  protein  

MMSYN1_0504  rRNA  small  subunit  S-­‐adenosylmethionine-­‐dependent  methyltransferase  

MMSYN1_0505   putative  liporotein  

   

69    

Table S11. Primers used in D serial construction. Primers used to amplify the CORE3 cassette for the deletion of gene clusters (column 1), and to detect junctions of the CORE3 cassette insertion and the cassette recycling (pop out).

Deleted  cluster  (MMSYN1)

 first  round  primer  set  1 second  round  primer  set  2 L  junction   R  junction   Pop  out

TTTTAATCCTCCAACCTATTAATATTTTAAATAAGATAAAACATTGTTGGTAGGGATAACAGGGTAATACGGATTAG

AGTATAAAACTTTTATCCTAACCGATTTTAAGTTTTATACAGGAGGAATTTTTTAATCCTCCAACCTATTAATAT

TCAACATATTCTGGTATGTCT

AAGTGTCACCATGAACGACA

TCAACATATTCTGGTATGTCT

CCAACAATGTTTTATCTTATTTAAAATATTAATAGGTTGGAGGATTAAAATCGGTACATAAATATATGTGATTCTG

CCAACAATGTTTTATCTTATTTAAAATATTAATAGGTTGGAGGATTAAAATCGGTACATAAATATATGTGATTCTG

CTTCGGAGGGCTGTCACC

ATATTCTACCGGTTTTTCTACC

ATATTCTACCGGTTTTTCTACC

AAATTATTTTTCCTTTTTTATAAACTCATAATTTTCTATTTTTTTAAGACATAGGGATAACAGGGTAATACGGATTAG

TTAGAAATAAAAATTGTATTTTTAAAATAAATATTTTAGGAAATGAAGCAAAATTATTTTTCCTTTTTTATAAACTCA

CACTAGCATTTTGACACCAAC

AAGTGTCACCATGAACGACA

CACTAGCATTTTGACACCAAC

TGTCTTAAAAAAATAGAAAATTATGAGTTTATAAAAAAGGAAAAATAATTTCGGTACATAAATATATGTGATTCT

TGTCTTAAAAAAATAGAAAATTATGAGTTTATAAAAAAGGAAAAATAATTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

AGCAGAAGTATCTCCTTGAG

AGCAGAAGTATCTCCTTGAG

TAATTCATTAGTCTCTAATTCTTATTAAAAGATTTAGAGATTTTTTATTTTAGGGATAACAGGGTAATACGGATTAG

CTAGAAATATATTAATATATCATTAAAAATAAAAATAGCATTTAAGATTTAATTCATTAGTCTCTAATTCTTATTAA

TGGTGTTGCTACTGAAATATG

AAGTGTCACCATGAACGACA

TGGTGTTGCTACTGAAATATG

AAATAAAAAATCTCTAAATCTTTTAATAAGAATTAGAGACTAATGAATTATCGGTACATAAATATATGTGATTCT

AAATAAAAAATCTCTAAATCTTTTAATAAGAATTAGAGACTAATGAATTATCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

ACTTAAAAAAGAACGACACCG

ACTTAAAAAAGAACGACACCG

TTATAAAGATTTATAATAATTAATATGCAAGACAAGAAAAATAAAGAAATTATAGGGATAACAGGGTAATACGGATTAG

TGCATTATATTGCAATAGAGATAAATTAACTTATTAAAAGAGATGATAAATTATAAAGATTTATAATAATTAATATGCAA

GCGAAATCAATTATTGTATAGAC

AAGTGTCACCATGAACGACA

GCGAAATCAATTATTGTATAGAC

TAATTTCTTTATTTTTCTTGTCTTGCATATTAATTATTATAAATCTTTATTCGGTACATAAATATATGTGATTCT

TAATTTCTTTATTTTTCTTGTCTTGCATATTAATTATTATAAATCTTTATTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

CTTGGTAAAGGAGTTTTTAAATC

CTTGGTAAAGGAGTTTTTAAATC

ATATTCACACCTATATTTTTAATCTGTTTTTAGACACATTAATACTAATTTAGGGATAACAGGGTAATACGGATTAG

TCAATGAACAACACTTTTATAGAATTATTTTATAAATATTTAGTTTTAAGATATTCACACCTATATTTTTAATCTGT

GATTTCATGACCATTACGAAG

AAGTGTCACCATGAACGACA

GATTTCATGACCATTACGAAG

AATTAGTATTAATGTGTCTAAAAACAGATTAAAAATATAGGTGTGAATATTCGGTACATAAATATATGTGATTCT

AATTAGTATTAATGTGTCTAAAAACAGATTAAAAATATAGGTGTGAATATTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

AGCAAGATATGCGATATGTAC

AGCAAGATATGCGATATGTAC

ATATAAAACTATTTACTCCCTAAAAGTTTATCTATTAACTCATAACACTTTAGGGATAACAGGGTAATACGGATTAG

AGCAAAAAAATGGACTAATATTTAAATAAAGCAAAAAGGAGAAAAAGATTATATAAAACTATTTACTCCCTAAAAG

GAAAAGAGCTATTAGAAGAGAC

AAGTGTCACCATGAACGACA

GAAAAGAGCTATTAGAAGAGAC

AAGTGTTATGAGTTAATAGATAAACTTTTAGGGAGTAAATAGTTTTATATTCGGTACATAAATATATGTGATTCT

AAGTGTTATGAGTTAATAGATAAACTTTTAGGGAGTAAATAGTTTTATATTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

GTTATAATTGGTACTAAATTAG

GTTATAATTGGTACTAAATTAG

AATTCACCTCATATTAGTATTAGAAAAATAATTAAAAATATTACATTTTTTAGGGATAACAGGGTAATACGGATTAG

TTGATTTTAAAAGTATAATAAAAATAGACAAATAGAAAGTTGGTATATAAAATTCACCTCATATTAGTATTAGAAA

GTATAGTTATTATAGCACCTATG

AAGTGTCACCATGAACGACA

GTATAGTTATTATAGCACCTATG

AAAAATGTAATATTTTTAATTATTTTTCTAATACTAATATGAGGTGAATTTCGGTACATAAATATATGTGATTCT

AAAAATGTAATATTTTTAATTATTTTTCTAATACTAATATGAGGTGAATTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

GTGAAAACTATGAATGTGATGC

GTGAAAACTATGAATGTGATGC

AAAATATCTTTCTAAAAATTAAAGGTCTTATTTATAAAAATTATAAATAATAGGGATAACAGGGTAATACGGATTAG

ACTGCATTAAATTTAAATAAAAATTATTTGCATATTAATTGTTGTCTTGGAAAATATCTTTCTAAAAATTAAAGGTC

AAGAAGAAAGTTCAACTGAAAC

AAGTGTCACCATGAACGACA

AAGAAGAAAGTTCAACTGAAAC

TTATTTATAATTTTTATAAATAAGACCTTTAATTTTTAGAAAGATATTTTTCGGTACATAAATATATGTGATTCT

TTATTTATAATTTTTATAAATAAGACCTTTAATTTTTAGAAAGATATTTTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

GTGGAGCATTACCAGATTC

GTGGAGCATTACCAGATTC

AATAGATTTAAAACAATTGAATTTGGTGATATATTAAAGGAAATGGGAATTAGGGATAACAGGGTAATACGGATTAG

TAAAAACATTAGATGAATAAACAAAAAACAGCTTAAAGCTGTTTTTTTTAAATAGATTTAAAACAATTGAATTTGGT

ACTACAAATGAAGCGATAACCT

AAGTGTCACCATGAACGACA

ACTACAAATGAAGCGATAACCT

ATTCCCATTTCCTTTAATATATCACCAAATTCAATTGTTTTAAATCTATTTCGGTACATAAATATATGTGATTCT

ATTCCCATTTCCTTTAATATATCACCAAATTCAATTGTTTTAAATCTATTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

GTACACTTTACCGACATGATC

GTACACTTTACCGACATGATC

TTTTGTTGTAGTAGGTTTAAATGGAACAATTTGAATTTTATTTTCTTTATTAGGGATAACAGGGTAATACGGATTAG

TTTTTTTTATTCAAAATCAACATAAAAATATTGTTTAAGGAGTATTAAATTTTTGTTGTAGTAGGTTTAAATGGA

TTGAGTTAGTAATTAATTGAAAGG

AAGTGTCACCATGAACGACA

TTGAGTTAGTAATTAATTGAAAGG

ATAAAGAAAATAAAATTCAAATTGTTCCATTTAAACCTACTACAACAAAATCGGTACATAAATATATGTGATTCT

ATAAAGAAAATAAAATTCAAATTGTTCCATTTAAACCTACTACAACAAAATCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

CAGCTGAAGAATATACTCCTG

CAGCTGAAGAATATACTCCTG

TTAAATAAAAAATGCAGGTGTAGAGGAGGAAAACCTGCATTTTTTTATTATAGGGATAACAGGGTAATACGGATTAG

AATAATTAGTGTAAATAAAAAAGTCATAAGTTAGACCTTATGACTTTTTATTAAATAAAAAATGCAGGTGTAGAG

CAAGGTAGTGAATATGATCAAG

AAGTGTCACCATGAACGACA

CAAGGTAGTGAATATGATCAAG

TAATAAAAAAATGCAGGTTTTCCTCCTCTACACCTGCATTTTTTATTTAATCGGTACATAAATATATGTGATTCT

TAATAAAAAAATGCAGGTTTTCCTCCTCTACACCTGCATTTTTTATTTAATCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

TATTATTGGTTCAAATGGTTGAG

TATTATTGGTTCAAATGGTTGAG

ATAGAAAAAAATGGATTAGTTGATAATCCATTTTTTAATTTTGTTTATTATAGGGATAACAGGGTAATACGGATTAG

CAAGATATACTACTAGTTTTACAAAAATATGATAAATAGAAAGGCTTAATATAGAAAAAAATGGATTAGTTGATAAT

AATACTGCTTGTCATTATTTGC

AAGTGTCACCATGAACGACA

AATACTGCTTGTCATTATTTGC

TAATAAACAAAATTAAAAAATGGATTATCAACTAATCCATTTTTTTCTATTCGGTACATAAATATATGTGATTCT

TAATAAACAAAATTAAAAAATGGATTATCAACTAATCCATTTTTTTCTATTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

CAAAGATCGTTTAGCTAAAGG

CAAAGATCGTTTAGCTAAAGG

TAAAAAATAAAAATATACTATTGGACACAAAAACTGAAATAATAAGTAATTTAGGGATAACAGGGTAATACGGATTAG

TTTTATTTAATATAAAAAAAACAAGATGATTTTTTCATCTTGTTTTTATTTAAAAAATAAAAATATACTATTGGACAC

GCTAAACAATTATTAGAGATTAG

AAGTGTCACCATGAACGACA

GCTAAACAATTATTAGAGATTAG

ATTACTTATTATTTCAGTTTTTGTGTCCAATAGTATATTTTTATTTTTTATCGGTACATAAATATATGTGATTCT

ATTACTTATTATTTCAGTTTTTGTGTCCAATAGTATATTTTTATTTTTTATCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

TTTTTTGAATAAGCAAATCCATC

TTTTTTGAATAAGCAAATCCATC

ATCATAGTCTTTAATTTATTTTTAATATTATTTTAAATAAATAACAAGCAAATAGGGATAACAGGGTAATACGGATTAG

TAGTACTTGTTTTTTTATTATTTAAAATATAGTATAAAAATAGGAAAAAAATCATAGTCTTTAATTTATTTTTAATATT

AGCTATGTATATGAGTATGGG

AAGTGTCACCATGAACGACA

AGCTATGTATATGAGTATGGG

TTTGCTTGTTATTTATTTAAAATAATATTAAAAATAAATTAAAGACTATGTCGGTACATAAATATATGTGATTCT

TTTGCTTGTTATTTATTTAAAATAATATTAAAAATAAATTAAAGACTATGTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

TGTCTACCATTTGAGATGACG

TGTCTACCATTTGAGATGACG

AACTTTAAAATAATTCCTTATAACTTAAATTATAACTTAATCGTAGTTACTAGGGATAACAGGGTAATACGGATTAG

ATCTATTGCTTTTGATATTTTAAAAAAAGTTTTTGTTAATAAAGCTTATTAACTTTAAAATAATTCCTTATAACTTAA

ACAAAGTTTTAGGACATGGTG

AAGTGTCACCATGAACGACA

ACAAAGTTTTAGGACATGGTG

GTAACTACGATTAAGTTATAATTTAAGTTATAAGGAATTATTTTAAAGTTTCGGTACATAAATATATGTGATTCT

GTAACTACGATTAAGTTATAATTTAAGTTATAAGGAATTATTTTAAAGTTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

TTGACATAAATATCCCTTTCTG

TTGACATAAATATCCCTTTCTG

GAACTGATGTAGAACATGTTGCAGATAAGTTAAAAGAAATTCTAAATTTAGGGATAACAGGGTAATACGGATTAG

AGATATGAGTTTAATAGTATTAGAAAATATTACACATCAAAACGGAGAAGGAACTGATGTAGAACATGTTGC

ACTGAACCAGACACCTTTCC

AAGTGTCACCATGAACGACA

ACTGAACCAGACACCTTTCC

AATTTAGAATTTCTTTTAACTTATCTGCAACATGTTCTACATCAGTTCCTTCGGTACATAAATATATGTGATTCT

AATTTAGAATTTCTTTTAACTTATCTGCAACATGTTCTACATCAGTTCCTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

GGTGCATAACGTAATCTAAATG

GGTGCATAACGTAATCTAAATG

ATCTAAAACTAAATTAAAGATTTATAATTTTAAATGTCTTTAGTTTAGTTTTAGGGATAACAGGGTAATACGGATTAG

AGGAGTTAATAAGTAATATAAGAACTTTTATAATTAATTTGAGGTGAAATATCTAAAACTAAATTAAAGATTTATAATT

TATACACTGTTCCATCAATATC

AAGTGTCACCATGAACGACA

TATACACTGTTCCATCAATATC

AAACTAAACTAAAGACATTTAAAATTATAAATCTTTAATTTAGTTTTAGATCGGTACATAAATATATGTGATTCT

AAACTAAACTAAAGACATTTAAAATTATAAATCTTTAATTTAGTTTTAGATCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

GTTATTATCTTATCATATTAAATGAC

GTTATTATCTTATCATATTAAATGAC

TTTAATAATTTTTAAAAAATAAAAATAATTTATATATGTTTTAATAGTTTAGATAGGGATAACAGGGTAATACGGATTAG

GGGATTAAATTATAATAGAAAATAAAAAAGGGCTTAAAAAAATTAAGCCCTTTAATAATTTTTAAAAAATAAAAATAATTTATA

GAACTTGGTGCTAATAAAGC

AAGTGTCACCATGAACGACA

GAACTTGGTGCTAATAAAGC

TCTAAACTATTAAAACATATATAAATTATTTTTATTTTTTAAAAATTATTTCGGTACATAAATATATGTGATTCT

TCTAAACTATTAAAACATATATAAATTATTTTTATTTTTTAAAAATTATTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

CAACATTTTAGCAGCCTCTC

CAACATTTTAGCAGCCTCTC

AATTTATTCTCCTAGATTATTATTACTTAAAATAATTATAACTTTTATTGTAGGGATAACAGGGTAATACGGATTAG

TCTTCATCTCAAATATTTACTATGTTTCAATCAAAATCAATATAAATCATAATTTATTCTCCTAGATTATTATTACT

CTCATCTCTTCATCAGTGTC

AAGTGTCACCATGAACGACA

CTCATCTCTTCATCAGTGTC

CAATAAAAGTTATAATTATTTTAAGTAATAATAATCTAGGAGAATAAATTTCGGTACATAAATATATGTGATTCT

CAATAAAAGTTATAATTATTTTAAGTAATAATAATCTAGGAGAATAAATTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

TTTGATGGTGACTCTAATAATAC

TTTGATGGTGACTCTAATAATAC

TTAAATGATAAAATCAAGGGATTTTTTTAAATATTTTAGAGATCATTGTTTAGGGATAACAGGGTAATACGGATTAG

AGCAAAAAAAATTACATCAAAATTGATGTAATTTTAATATTATTTATTTATTAAATGATAAAATCAAGGGATTTT

ATAAGAAATACTAGAATAGTGTC

AAGTGTCACCATGAACGACA

ATAAGAAATACTAGAATAGTGTC

AACAATGATCTCTAAAATATTTAAAAAAATCCCTTGATTTTATCATTTAATCGGTACATAAATATATGTGATTCT

AACAATGATCTCTAAAATATTTAAAAAAATCCCTTGATTTTATCATTTAATCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

TGCTAGTAGTTTTCTAATATATC

TGCTAGTAGTTTTCTAATATATC

CAAATTACTAAAAAAGAGCTATGAAGCTCTTTTTTTAATGAATCCTTATTAGGGATAACAGGGTAATACGGATTAG

TTTCTAAAAATAATTTTTAGACAATGTATTGTGTTCTTTTTAAAAACTAACAAATTACTAAAAAAGAGCTATGAAG

CAGATCAATCTAATAACAATTCC

AAGTGTCACCATGAACGACA CTTCGGAGGGCTGTCACC

ATAAGGATTCATTAAAAAAAGAGCTTCATAGCTCTTTTTTAGTAATTTGTTCGGTACATAAATATATGTGATTCT

ATAAGGATTCATTAAAAAAAGAGCTTCATAGCTCTTTTTTAGTAATTTGTTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

TGTGTTTTAAATCGTTTTATGCT

TGTGTTTTAAATCGTTTTATGCT

ATATAACCTACCCCTTAAATTATCTATTTTAATAAAAAACTTATTATATCTAGGGATAACAGGGTAATACGGATTAG

GGCAGCAGCCACTGGTAACAGGATTAGCAGAGATCTAGGTTTTTTCTATTATATAACCTACCCCTTAAATTATCT

GTAACTATCGTCTTGAGTCC

AAGTGTCACCATGAACGACA

GTAACTATCGTCTTGAGTCC

GATATAATAAGTTTTTTATTAAAATAGATAATTTAAGGGGTAGGTTATATTCGGTACATAAATATATGTGATTCT

GATATAATAAGTTTTTTATTAAAATAGATAATTTAAGGGGTAGGTTATATTCGGTACATAAATATATGTGATTCT

CTTCGGAGGGCTGTCACC

AGATACAATAAAGATATAATAAGAC

AGATACAATAAAGATATAATAAGAC

D  serial  deletion  amplification  of  the  core3  cassette  (2  rounds  of  PCR) junction    PCR

0550  -­‐0591

0698-­‐0703

0673-­‐0676

0594-­‐0598

0019-­‐0024

0455-­‐0474

0337-­‐0343

0272-­‐0276

0318-­‐0324

0204-­‐0212

0118-­‐0125

0084-­‐0088

0889-­‐0904

0711-­‐0716

0309-­‐0313

0854-­‐0857  and  0858

0241-­‐0246

0734-­‐0770

0860-­‐0869

0170-­‐0179  and  0187-­‐0194  

0841-­‐0850

0180-­‐0186

   

70    

Table S12. Primers used in NotI site construction. Primers used to amplify the cassette for engineering NotI sites, to detect junctions of the CORE3 cassette insertion, and the cassette recycling (pop out). 208 bp was inserted into the location of NotI (Fig, S9). The CORE cassette for engineering NotI site at the NotI-8D location was PCR-amplified by only 1 round (30 cycles) and the 305 bp containing the 208 bp was also amplified by 1 round (30 cycles). 5 to 9 nucleotides (lowercase) were embedded into primers for the NotI site engineering.

 first  round  primer  set  1 second  round  primer  set  2 L  junction   R  junction   Pop  out

ATTAGTCTCTAATTCTTATTAAAAGATTTAGAGATTTTTTATTTACTTACTAGGGATAACAGGGTAATACGGATTAG CTATAAGCTTCAAATTCACAATAAAAGAACAAAATAAAAAATAATAATTCgcggccgcATTAGTCTCTAATTCTTATTAAAAGAT GGAAGTGGAACTGTAAAATC AAGTGTCACCATGAACGACA GGAAGTGGAACTGTAAAATC

GTAAGTAAATAAAAAATCTCTAAATCTTTTAATAAGAATTAGAGACTAATTCGGTACATAAATATATGTGATTCT GTAAGTAAATAAAAAATCTCTAAATCTTTTAATAAGAATTAGAGACTAATTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC GTGATCCAAACAGCAAATTTG GTGATCCAAACAGCAAATTTG

AAAACACTGGAATTCCATATATTTTTTTCTAAATTACTAAAAAACAATCTTAGGGATAACAGGGTAATACGGATTAG TTTTATATTAGTAAATCTAGTAATAATACAAATTTCTAAAAACTAAAGAAgcggccgcAAAACACTGGAATTCCATATATTTT CTTTATGAGCGACATCAGTC AAGTGTCACCATGAACGACA CTTTATGAGCGACATCAGTC

AGATTGTTTTTTAGTAATTTAGAAAAAAATATATGGAATTCCAGTGTTTTTCGGTACATAAATATATGTGATTCT AGATTGTTTTTTAGTAATTTAGAAAAAAATATATGGAATTCCAGTGTTTTTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC AATTTGGCTCTGATCAACACC AATTTGGCTCTGATCAACACC

ATATTCTGGACTAAAATCCAGGAAATGGGGGTTTTTATATATCCAAATTATAGGGATAACAGGGTAATACGGATTAG TAAATTGCATTATTCTTTAAATTATTTGGTATAGTGGTATCACGAATCCCgcggccgcATATTCTGGACTAAAATCCAGGA TAGAGTATAACTTGAACTAGAG AAGTGTCACCATGAACGACA TAGAGTATAACTTGAACTAGAG

TAATTTGGATATATAAAAACCCCCATTTCCTGGATTTTAGTCCAGAATATTCGGTACATAAATATATGTGATTCT TAATTTGGATATATAAAAACCCCCATTTCCTGGATTTTAGTCCAGAATATTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC CTGATGTCGCTCATAAAGTG CTGATGTCGCTCATAAAGTG

CGCACCTCTGGCTTTTATTTTGATCAAATATTTATAGAATATTTTTATAATAGGGATAACAGGGTAATACGGATTAG TGTCGGTAAAGTGTACGTCACCCCGGCCAAACTATCGCCAGATAAGTGTAgcggcCGCACCTCTGGCTTTTATTTTG GGTGATATATTAAAGGAAATGG AAGTGTCACCATGAACGACA GGTGATATATTAAAGGAAATGG

TTATAAAAATATTCTATAAATATTTGATCAAAATAAAAGCCAGAGGTGCGTCGGTACATAAATATATGTGATTCT TTATAAAAATATTCTATAAATATTTGATCAAAATAAAAGCCAGAGGTGCGTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC CTAGCTGACACGAATCTTAC CTAGCTGACACGAATCTTAC

CGCAAATAGATTTAAAACAATTGAATTTGGTGATATATTAAAGGAAATGGGAATTAGGGATAACAGGGTAATACGGATTAG GTTTTACCTTACTAAAAAAAGATATATTAGAGATTTTTACTAAGTTTCAAgcggccgcaAATAGATTTAAAACAATTGAA GAAATATTTGTGCGCATGAAG AAGTGTCACCATGAACGACA GAAATATTTGTGCGCATGAAG

ATTCCCATTTCCTTTAATATATCACCAAATTCAATTGTTTTAAATCTATTTCGGTACATAAATATATGTGATTCT ATTCCCATTTCCTTTAATATATCACCAAATTCAATTGTTTTAAATCTATTTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC GGTGACGTACACTTTACCG GGTGACGTACACTTTACCG

TTGACTGTGCATAGTTCGTTCATGCGCTCGTTGCGTGATAAACTAGGCAGTAGGGATAACAGGGTAATACGGATTAG TTGACTGTGCATAGTTCGTTCATGCGCTCGTTGCGTGATAAACTAGGCAGTAGGGATAACAGGGTAATACGGATTAG GATAGTTTAGGCTCGTTGCA AAGTGTCACCATGAACGACA GATAGTTTAGGCTCGTTGCA

CTGCCTAGTTTATCACGCAACGAGCGCATGAACGAACTATGCACAGTCAATCGGTACATAAATATATGTGATTCT CTAGCACTAGACAGCCAGCAATATCAGCAATTAAATTATCAGTTGTGAAGcggccgCTGCCTAGTTTATCACGCAACG CTTCGGAGGGCTGTCACC AGTTTCATTATCAAGCACGTG AGTTTCATTATCAAGCACGTG

ACGTAACAACCATATAAGTTAGATTTAATGCCCCTGACTGAACGCTCGTTTAGGGATAACAGGGTAATACGGATTAG CAATAGCTATTGCTGTGCATAGGCCTATAGTGGCTGTAACTAGTGATATCagcggccgcACGTAACAACCATATAAGTTAGATT GTGCATACAACGTCATGTGA AAGTGTCACCATGAACGACA GTGCATACAACGTCATGTGA

AACGAGCGTTCAGTCAGGGGCATTAAATCTAACTTATATGGTTGTTACGTTCGGTACATAAATATATGTGATTCT AACGAGCGTTCAGTCAGGGGCATTAAATCTAACTTATATGGTTGTTACGTTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC CTTGCTAAGCGGAACTTATC CTTGCTAAGCGGAACTTATC

GcggccgcTAAAAATTTTGATATAAAATTTATTTTAAAAATTATTCATGAAAATTATGTAGGGATAACAGGGTAATACGGATTAG AAATAACACTATTTTTTCTATTGATTACTAGTTTTTTTTAATATTTATTAGcggccgcTAAAAATTTTGATAT CAACTTATACTTGCTTAAAAACAC AAGTGTCACCATGAACGACA CAACTTATACTTGCTTAAAAACAC

CATAATTTTCATGAATAATTTTTAAAATAAATTTTATATCAAAATTTTTATCGGTACATAAATATATGTGATTCT CATAATTTTCATGAATAATTTTTAAAATAAATTTTATATCAAAATTTTTATCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC GACAACCTTTTAGATTATTTGG GACAACCTTTTAGATTATTTGG

AAAACTCTATGGTTTTTGATAAAAATTCTAAAAATAAACTTTTAAATGAGTAGGGATAACAGGGTAATACGGATTAG CACATTTATATTATATTATTTTAAAAAATAACAAAATAAACTAGTGTATAgcggccgcAAAACTCTATGGTTTTTGATAAAAATT ATCCATTACTGTAAGATCGG AAGTGTCACCATGAACGACA ATCCATTACTGTAAGATCGG

CTCATTTAAAAGTTTATTTTTAGAATTTTTATCAAAAACCATAGAGTTTTTCGGTACATAAATATATGTGATTCT CTCATTTAAAAGTTTATTTTTAGAATTTTTATCAAAAACCATAGAGTTTTTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC TGTTTTTAAGCAAGTATAAGTTG TGTTTTTAAGCAAGTATAAGTTG

CGCAATTTCTAATATGTCATCTAAAACATCTTTTGAAAGATTAGATAAAAATTTAGGGATAACAGGGTAATACGGATTAG AAATCAACAAAATCAAGTTTAGTTAAATCAACTTCATATTCATTTTTATAagcggccgcAATTTCTAATATGTCATCTAAA CTAAGTAACTAGTGACTTTATC AAGTGTCACCATGAACGACA CTAAGTAACTAGTGACTTTATC

AATTTTTATCTAATCTTTCAAAAGATGTTTTAGATGACATATTAGAAATTTCGGTACATAAATATATGTGATTCT AATTTTTATCTAATCTTTCAAAAGATGTTTTAGATGACATATTAGAAATTTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC TTCAGATTCATCATATATGTCAG TTCAGATTCATCATATATGTCAG

CCGCCATGATCATAAATTTCAATTTGATTATCAAAACTATTTTCAATATTGTTAGGGATAACAGGGTAATACGGATTAG TTAGTTATATATTCATTTTCAAATTCAATAATTGATTTTGTATGATGTGAgcggccGCCATGATCATAAATTTCAAT TAAAGATCAATGGGTTGATAAG AAGTGTCACCATGAACGACA TAAAGATCAATGGGTTGATAAG

ACAATATTGAAAATAGTTTTGATAATCAAATTGAAATTTATGATCATGGCTCGGTACATAAATATATGTGATTCT ACAATATTGAAAATAGTTTTGATAATCAAATTGAAATTTATGATCATGGCTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC AGATGTTTTAGATGACATATTAGA AGATGTTTTAGATGACATATTAGA

gcggccgcACTGAATTATTTTTCAAATTGTTAAATTATTTTCAAATCATTATCTATTATAGGGATAACAGGGTAATACGGATTAG ATATATTAGAAAACTACTAGCACTTTTTTAAAAAATTTTTAATTTTAATAgcggccgcACTGAATTATTTT TACTTTTAGTCTCATAAATCTAAG AAGTGTCACCATGAACGACA TACTTTTAGTCTCATAAATCTAAG

TAATAGATAATGATTTGAAAATAATTTAACAATTTGAAAAATAATTCAGTTCGGTACATAAATATATGTGATTCT TAATAGATAATGATTTGAAAATAATTTAACAATTTGAAAAATAATTCAGTTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC GCTTTATTTTGATCCATAATTCAC GCTTTATTTTGATCCATAATTCAC

gcggccgcTTAAATGATAAAATCAAGGGATTTTTTTAAATATTTTAGAGATCATTGTTTAGGGATAACAGGGTAATACGGATTAG TCGTAAAAATTGAAATAATAATCCAACTACTAGTCCAATCAAAAAAAATAgcggccgcTTAAATGATAAAATCA AACACCAACATTACATTCTTTTG AAGTGTCACCATGAACGACA AACACCAACATTACATTCTTTTG

AACAATGATCTCTAAAATATTTAAAAAAATCCCTTGATTTTATCATTTAATCGGTACATAAATATATGTGATTCT AACAATGATCTCTAAAATATTTAAAAAAATCCCTTGATTTTATCATTTAATCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC GTGCTAGTAGTTTTCTAATATATC GTGCTAGTAGTTTTCTAATATATC

gcTAACATAGGATCAACTACTATTACATAACTTTTATCAATATCTTTAGTAGTAGGGATAACAGGGTAATACGGATTAG CCTCATTGTTTAACGATATCTATTGCTTTACATGCACTTCCACCAGTTGCggccgcTAACATAGGATCAACTACTAT CTGGATGTTGTTCTTGCAGC AAGTGTCACCATGAACGACA CTGGATGTTGTTCTTGCAGC

CTACTAAAGATATTGATAAAAGTTATGTAATAGTAGTTGATCCTATGTTATCGGTACATAAATATATGTGATTCT CTACTAAAGATATTGATAAAAGTTATGTAATAGTAGTTGATCCTATGTTATCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC CCAGTTGCAAAAACTACAGG CCAGTTGCAAAAACTACAGG

GCATCTCCTAATCCTGGAACGATATAACCTTTTTCATTTAATTTTTCATCTAGGGATAACAGGGTAATACGGATTAG ACGTATCATTTATAAATTTGAATTATTTTGTTCCAAAAATTCTATCTCCAgcggccGCATCTCCTAATCCTGGAACG AATCTGCTTTAACATTGTGAATG AAGTGTCACCATGAACGACA AATCTGCTTTAACATTGTGAATG

GATGAAAAATTAAATGAAAAAGGTTATATCGTTCCAGGATTAGGAGATGCTCGGTACATAAATATATGTGATTCT GATGAAAAATTAAATGAAAAAGGTTATATCGTTCCAGGATTAGGAGATGCTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC AGGCTGCAAGAACAACATC AGGCTGCAAGAACAACATC

cCTATTTAATGTTTTTAATAAGTTCAATAATTGACTGAATGATAAATTTAATAGGGATAACAGGGTAATACGGATTA TTCATGTTGATTTAGAATATTGC AAGTGTCACCATGAACGACA TTCATGTTGATTTAGAATATTGC

TTAAATTTATCATTCAGTCAATTATTGAACTTATTAAAAACATTAAATAGTCGGTACATAAATATATGTGATTCT CTTCGGAGGGCTGTCACC TAAAGACGATTTATTAGAGTTACT TAAAGACGATTTATTAGAGTTACT

ACTAGAAATATATTAATATATCATTAAAAATAAAAATAGCATTTAAGATTATTAGTCTCTAATTCTTATTAAAAG

TAAATTTATCATTCAGTCAATTATTGAACTTATTAAAAACATTAAATAGgcggccGCCTCTTTAGATTTTAGTGATC

NotI-­‐5U

Not  I  site  engineering

amplification  of  the  core3  cassette  (2  rounds  of  PCR) junction    PCR

NotI-­‐1U

NotI-­‐1D

NotI-­‐2U

NotI-­‐2D

NotI-­‐3U

NotI-­‐3D

NotI-­‐4U

NotI-­‐4D

NotI-­‐8D

305  bp

NotI-­‐5D

NotI-­‐6U

NotI-­‐6D

NotI-­‐7U

NotI-­‐7D

NotI-­‐8U

   

71    

Landing  pad  insertion  

primer    set   L  junction   R    junction  

1  

ACTAGAAATATATTAATATATCATTAAAAATAAAAATAGCATTTAAGATTTACCGTTCGTATAAGAAACCA   TTCATGTTGATTTAGAATATTGC   ACTTTAGTGACGAGATTGAGT  

AGATTGTTTTTTAGTAATTTAGAAAAAAATATATGGAATTCCAGTGTTTTTACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   AATTTGGCTCTGATCAACACC  

2  

TAAATTGCATTATTCTTTAAATTATTTGGTATAGTGGTATCACGAATCCCTACCGTTCGTATAAGAAACCA   TCCATATACTATTGTAGATTTTG   ACTTTAGTGACGAGATTGAGT  

TTATAAAAATATTCTATAAATATTTGATCAAAATAAAAGCCAGAGGTGCGTACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   TACCTAGCTGAACTTATACC  

3  

GTTTTACCTTACTAAAAAAAGATATATTAGAGATTTTTACTAAGTTTCAATACCGTTCGTATAAGAAACCA   GAATATAGCGACTATGATGG   ACTTTAGTGACGAGATTGAGT  

CTAGCACTAGACAGCCAGCAATATCAGCAATTAAATTATCAGTTGTGAAGTACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   GTTTCATTATCAAGCACGTG  

4  

CAATAGCTATTGCTGTGCATAGGCCTATAGTGGCTGTAACTAGTGATATCTACCGTTCGTATAAGAAACCA   GATACAACGTTGCTGGCTG   ACTTTAGTGACGAGATTGAGT  

CATAATTTTCATGAATAATTTTTAAAATAAATTTTATATCAAAATTTTTATACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   GAACTGTAGATCAGCAAATTG  

5  

CACATTTATATTATATTATTTTAAAAAATAACAAAATAAACTAGTGTATATACCGTTCGTATAAGAAACCA   TACTGTAAGATCGGCAAATGA   ACTTTAGTGACGAGATTGAGT  

AATTTTTATCTAATCTTTCAAAAGATGTTTTAGATGACATATTAGAAATTTACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   AATGAGCAATTTCAGATTCATC  

6  

TTAGTTATATATTCATTTTCAAATTCAATAATTGATTTTGTATGATGTGATACCGTTCGTATAAGAAACCA   TAAAGATCAATGGGTTGATAAG   ACTTTAGTGACGAGATTGAGT  

TAATAGATAATGATTTGAAAATAATTTAACAATTTGAAAAATAATTCAGTTACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   GCTCCAACTAAAATCAGATTG  

7  

TCGTAAAAATTGAAATAATAATCCAACTACTAGTCCAATCAAAAAAAATATACCGTTCGTATAAGAAACCA   ATTTTGTTTAATCATTGGACTGA   ACTTTAGTGACGAGATTGAGT  

CTACTAAAGATATTGATAAAAGTTATGTAATAGTAGTTGATCCTATGTTATACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   GCTCATGTTGGTTTATATAGAG  

8  

ACGTATCATTTATAAATTTGAATTATTTTGTTCCAAAAATTCTATCTCCATACCGTTCGTATAAGAAACCA   TGGTGTTGCTACTGAAATATG   ACTTTAGTGACGAGATTGAGT  

ATAATTACTCAAAAAATCCATATCTTGGAATTTTAGAAAATGTTATTAGATACCGTTCGTATAGCATACAT   GTTTGCTTTTCGTGCACTAAACA   GCTCATGTTGGTTTATATAGAG  

Table S13. Primers used to amplify the landing pad cassette for landing pad strain constructions and to detect cassette insertion junctions.

   

72    

Table S14: Primers used to amplify the CORE6 cassette for the deletions of 39 gene and gene clusters and to detect insertion junctions.

gene  or  gene  cluster Primer  set  1 Primer  set  2 L  junction R  JunctionGAATTGGTTAAAGTAAAGGATAACTATTAGGGATAACAGGGTAATACGGATTAG ATACAAAATTAATTCATCTACTAGAATTGGTTAAAGTAAAGGATAAC ATTACTATCATTAATGAAGATGG GTTGCATTCGATTCCTGTTGCGCTCTAATCTAGGTTTTTTTAGTGTTTATATAAGTGGCCAGCCATTACGCTCG CCGCCTACATACCTCGCTCTAATCTAGGTTTTTTTAG CTTCGGAGGGCTGTCACC TCAGCAGAGCGCAGATACGCAGAGATCTAGGTTTTTTCTATTTAGGGATAACAGGGTAATACGGATTAG GGCAGCAGCCACTGGTAACAGGATTAGCAGAGATCTAGGTTTTTTCTA TCGTTCGCTCCAAGCTGG GTTGCATTCGATTCCTGTTGATAGATAATTTAAGGGGTAGGTTATATGGCCAGCCATTACGCTCG GATATAATAAGTTTTTTATTAAAATAGATAATTTAAGGGGTAGGTTAT CTTCGGAGGGCTGTCACC GATATAATAGTGATATAGATGATACTAAGTATTCAGGAGTAAATTTATAGGGATAACAGGGTAATACGGATTAG TGCATACTATAAAAAATTGTTTTTCAAATAAGTATTCAGGAGTAAATTTATAG GCTGAACCAAGTACAATGC GTTGCATTCGATTCCTGTTGCAAAAAACCAAGATTTATAACTCTTGGTTTTTGGCCAGCCATTACGCTCG GACAAATAATAATATAAACAAAAAACCAAGATTTATAACTCTTG CTTCGGAGGGCTGTCACC TTCTGGGAAAATTATTGATTTAGCCTTTTTTTAAAGTGGTGATTTTTAGGGATAACAGGGTAATACGGATTAG CCCTATGCTGGAAGGAGACTATGTCTCCTTTTTTTAAAGTGGTGATTTTTAG CACATCCAAATATAAATAATGACG GTTGCATTCGATTCCTGTTGTAAATAAAATTAAAAAGCATTATTAGGGCCAGCCATTACGCTCG ATAATGTTAAAGATAAAGTTATTTTAAATAAAATTAAAAAGCATTATTAGG CTTCGGAGGGCTGTCACC GTATAAATCAATAACAACAATTGCAGTCTTATTCAATTAAGTAGATATAGGGATAACAGGGTAATACGGATTAG CGGCGTTATAATTATATTATATTTTTAAGTCTTATTCAATTAAGTAGATATAG CCGCAATACAGAACACTTTTC GTTGCATTCGATTCCTGTTGCAAAAAAACAAGCCGTGGCTTGTTTTGATAGGCCAGCCATTACGCTCG ATTATAAGTAATTCTTAAAACAAAAAAACAAGCCGTGGCTTG CTTCGGAGGGCTGTCACC GATTACGCTTGTTTAATCTTTGCTAAAACCAAAATGAGGATAGAAATAGGGATAACAGGGTAATACGGATTAG AACTAATAACTTTTAAAAGTTTTTTTCTAAAACCAAAATGAGGATAGAAA GCTAACGAGTCACAATGACC GTTGCATTCGATTCCTGTTGGTTTGTAAACAAAAACTAAAGAAAGAGAATAATGGCCAGCCATTACGCTCG CTAAATAAATCTATTTAGTTTGTAAACAAAAACTAAAGAAAG CTTCGGAGGGCTGTCACC CGCAATTGCATACTTGTATGTCTCCTAATTTAAAATATATTTTAGGGATAACAGGGTAATACGGATTAG TTCAAATATTTGTCTGATTTCCATAATATCTCCTAATTTAAAATATATTTTAGG TCTTGCTTGTAATGCTTGTTC GTTGCATTCGATTCCTGTTGATAATTTTAAAAAGAAAGATTATTGGCCAGCCATTACGCTCG GAAAAAATTCTTTTAAATAACAAATAATAATTTTAAAAAGAAAGATTATTGGC CTTCGGAGGGCTGTCACC CTGCTATGATTGCTAAATTAGC

GATGGTTGAGTAATCTTATATTTATAGGGATAACAGGGTAATACGGATTAG AAAAGAATACCCACAATAGTTTTTTAGATGGTTGAGTAATCTTATATTTATAG GAAAATTCATCACAACTTGACTC GTTGCATTCGATTCCTGTTGATTTATATTTAAATAAAGGAGAAATGGCCAGCCATTACGCTCG AACTCTATAGTAAAATAAAATATATATTTATATTTAAATAAAGGAGAAATGG CTTCGGAGGGCTGTCACC CAGGAGCAACTACTGTATC

TATCCTAATATAAAATTTTATAGGGATAACAGGGTAATACGGATTAG TTTATTGGTTTAACAATTATGATATTTTAATATCCTAATATAAAATTTTATAGGGA TGCTTTATTTGCTAATCTTGAC GTTGCATTCGATTCCTGTTGGGAGGTTGTGATGCTAAAACGATTTAAGGCCAGCCATTACGCTCG ATAATAAAATTAATGATAAATATGGAGGTTGTGATGCTAAAACG CTTCGGAGGGCTGTCACC TTATACACTTCATTACAACCATC

CATTATAATAACGATGAATTCTAGTAGGGATAACAGGGTAATACGGATTAG TTAATTTCTTTAGTGTTGAACAAAAACATTATAATAACGATGAATTCTAGTA GCTAAAAACTTTGATGCTGAAAC GTTGCATTCGATTCCTGTTGGTTATAAGGAATTATTTTAAAGTTGGCCAGCCATTACGCTCG GTAACTACGATTAAGTTATAATTTAAGTTATAAGGAATTATTTTAAAGTTGG CTTCGGAGGGCTGTCACC TTCTATTCTTGACATAAATATCC

GCTTAATTTATTAAGTTATAAAATTTAGGGATAACAGGGTAATACGGATTAG TCATCAATTGATGAGCTTTTCTTTTGCTTAATTTATTAAGTTATAAAATTTAGG TAGAAAAATTGATTTAGTTGCTG GTTGCATTCGATTCCTGTTGGAATTTTTTAAAAAAGAATATTGGCCAGCCATTACGCTC TTTTTTCAAGTAATTCTTATAAAATTTTGAATTTTTTAAAAAAGAATATTGGCC CTTCGGAGGGCTGTCACC CTTAGAAAAGCAAAATGACTTAC

CTTTAAAAGAAAGGGCTTTTAATATAGGGATAACAGGGTAATACGGATTAG ATGAGATAAAAAACCAAGACTAAAAACTTTAAAAGAAAGGGCTTTTAATATAG TTAGTGGTTTAATATATTAGTTGG GTTGCATTCGATTCCTGTTGCCTTTCTAAATATTTATTATGGCCAGCCATTACGCTCG CAGCAAATTTAACTTTGAACATATCTTTTTCCTTTCTAAATATTTATTATGGCC CTTCGGAGGGCTGTCACC AGCAGGTATTTCACTGTTTAC

ATTAATAAAAGAAAGTAATAATAGGGATAACAGGGTAATACGGATTAG AACTTAGCTTAAAAAGCTAAGTTTTTAATATTAATAAAAGAAAGTAATAATAGGGA TAGTGAAGAATCAAAAATTCCAG GTTGCATTCGATTCCTGTTGAATCAATTTGAATTATAATAATGGCCAGCCATTACGCTCG ATCTACTAAGATAAAAAAACAACTTTATAATCAATTTGAATTATAATAATGGCC CTTCGGAGGGCTGTCACC CCATCACTTGCTGCAATTC

TAATAGTAAGAGGTATTAAATAGGGATAACAGGGTAATACGGATTAG TAATTTAAATAACAAGTTTATTAGATATTTTAATAGTAAGAGGTATTAAATAGG ACAACATGAACAAGCAAGAAG GTTGCATTCGATTCCTGTTGAATCAAGCTTAAGCTTGATTTTTGGCCAGCCATTACGCTCG AAATAAAAATTAAAAACTCATAATAAAAATCAAGCTTAAGCTTGATTTTTG CTTCGGAGGGCTGTCACC GAAGTTAAAGGTGAATTTGTAAG

GCAAAAAGGAGAAAAAGATTTAGGGATAACAGGGTAATACGGATTAG AGCAAAAAAATGGACTAATATTTAAATAAAGCAAAAAGGAGAAAAAGATTTAGG GAAAAGAGCTATTAGAAGAGAC GTTGCATTCGATTCCTGTTGCTTTTAGGGAGTAAATAGTTTTATATGGCCAGCCATTACGCTCG AAGTGTTATGAGTTAATAGATAAACTTTTAGGGAGTAAATAGTTTTATAT CTTCGGAGGGCTGTCACC ATACCAGTTGTTATAATTGGTAC

ATATAGAAAAGAGCATAAGAATAGGGATAACAGGGTAATACGGATTAG TTACTAGTAATATTTTATTTTAATATATAATATAGAAAAGAGCATAAGAATAGG GTTAGAATATAAGCTAAACCTG GTTGCATTCGATTCCTGTTGCAATAGCTTTTTTTATAGGAGAGTAATGGCCAGCCATTACGCTCG AAATAATATAGCTATTGTCATTTCAATAGCTTTTTTTATAGGAGAGTA CTTCGGAGGGCTGTCACC ATGAATCACCAATAGCTAAAGC

CTCATAATCTTTACCTACTTTTAGGGATAACAGGGTAATACGGATTAG TTGGTAATAAATCAACTTGTTGTTTTAAACTCATAATCTTTACCTACTTTTAG AGTAATTTCTCTAACTAGTCTTG GTTGCATTCGATTCCTGTTGTGGATTATAAAATTGGTACAAAATTGGCCAGCCATTACGCTCG AGATATGCCAGTTATATAATTAAATTGGATTATAAAATTGGTACAAAATTG CTTCGGAGGGCTGTCACC AGATAAATCATAATCTGGTTTATC

CTAGTGAGGCAATGTAAATAATAGGGATAACAGGGTAATACGGATTAG TAAAAAAGCTATAAAGTTAGAAAATGCCTCTAGTGAGGCAATGTAAATAATAG CAGTAACATTTGGTGGAGG GTTGCATTCGATTCCTGTTGGATTCAACTTATGTTATTGGTGTTGAAAGAGGCCAGCCATTACGCTCG ACCTTGAAATTTTTAACAACGATTCAACTTATGTTATTGGTGTTG CTTCGGAGGGCTGTCACC TGGTAGTTATTTATTTGGTAGC

GGCATTATAAAACCTCTAATTAGGGATAACAGGGTAATACGGATTAG TTATTTATTCCTAAATCTTTTTTAGTTTTTGGCATTATAAAACCTCTAATTAGG AGAAGCATCTAAATCAAGTGC GTTGCATTCGATTCCTGTTGTTTCTAATACTTTAATAGGAGATGATTAAAGGCCAGCCATTACGCTCG AGTTAATTTTAAAAACTTTTTTTCTAATACTTTAATAGGAGATGA CTTCGGAGGGCTGTCACC ACTTTAACTTTCATATATAACCTC

GATTTTTTCATCTTGTTTTTTAGGGATAACAGGGTAATACGGATTAG AAGTTTTATTTAATATAAAAAAAACAAGATGATTTTTTCATCTTGTTTTTTAGGG AGATTAGTTTAATTGATCAAGATC GTTGCATTCGATTCCTGTTGAAGACTTTCTAAGAAAGTCTTTTTGGCCAGCCATTACGCTCG ATAATTTATAGTAATTAATACAAAAAAAGACTTTCTAAGAAAGTCTTTTTG CTTCGGAGGGCTGTCACC ATCAACAAATCTCTGATTTAAAAC

CACACTTATAAAAGTATGTATAGGGATAACAGGGTAATACGGATTAG ATTAGGAATAGATCTAAAATTAAAATAGTACACACTTATAAAAGTATGTATAGG TTTATCTCAAGGAAATATCAATGA GTTGCATTCGATTCCTGTTGTTTAGAAAGTTTAGAATACCAAAGGCCAGCCATTACGCTCG ATAATTTTTAGTTTTATATAAAATACTTTTAGAAAGTTTAGAATACCAAAGG CTTCGGAGGGCTGTCACC TCTCTGGTAAACTACTAACTC

CCAAATTAGAGGTAATTAATTAGGGATAACAGGGTAATACGGATTAG ATTATGAATAAGTTAAACTCAATGAAAAATCCAAATTAGAGGTAATTAATTAGG GAATTACTAATTGGTGAAGAATAC GTTGCATTCGATTCCTGTTGGCATAAATAATAAGATTTAAAACTAGGCCAGCCATTACGCTCG ATAAAAAATCTTATTATTTAATAAAGCATAAATAATAAGATTTAAAACTAGG CTTCGGAGGGCTGTCACC AACCTTAACTCTATTATTTACACT

TTAGAGAGAAAAAATAATTAGGGATAACAGGGTAATACGGATTAG TGGTCTTGATAGTATTCCAATTAATTTAATTTTTAGAGAGAAAAAATAATTAGGGAT AAGTTCAAGCTTATATTCCAAC GTTGCATTCGATTCCTGTTGCAACTTAAACCAGTCAAACACCTTTAGGCCAGCCATTACGCTCG AAATTATTTCTCTTTTCTACTTTTCAACTTAAACCAGTCAAACACC CTTCGGAGGGCTGTCACC GATTCTATTAACAACTTTTTCTGC

TTATAAAAGGCTATAAAGTAGGGATAACAGGGTAATACGGATTAG TTAATACAATATTTAGTAATTAGAAAAATAATTTATAAAAGGCTATAAAGTAGGGAT AAGTAAATCAAGATAGAAATGATC GTTGCATTCGATTCCTGTTGGAGTTGTAGATGGCATCATACCTGGTTGGCCAGCCATTACGCTCG AGGATATGCTGATGGGGTTGATGGAGTTGTAGATGGCATCATAC CTTCGGAGGGCTGTCACC TAGCTAAACTAATTACTAATAGTG

TTAATAGATTGTGGTGATTTTTAGGGATAACAGGGTAATACGGATTAG GGGTTATAGATAAAATCTCGTCATTTTTTTTAATAGATTGTGGTGATTTTTAGGG GCCACAGAGACGAATAAAGAG GTTGCATTCGATTCCTGTTGTTAACTTGTTCAGTTAGTTCATTTAGGCCAGCCATTACGCTCG TTTTTAATCTTGTTTAATTAGTTTTTTAACTTGTTCAGTTAGTTCATTTAG CTTCGGAGGGCTGTCACC TCCATATTATCTATTTTCTTTCTGC

GATATTAGTTCATTATGATAGATTAGGGATAACAGGGTAATACGGATTAG AATCCAGAAGATAATAAACAAGGTAATGATATTAGTTCATTATGATAGATTAG GATATGTTACTGAAGATATTTTAG GTTGCATTCGATTCCTGTTGATAAATAGTGATAAAAATTGGCCAGCCATTACGCTCG AAAAATTAACTTATTTTTAAAAATATAAAATATAAATAGTGATAAAAATTGGCCAG CTTCGGAGGGCTGTCACC TCTAGTTGTCCTATAATGTTTAC

ATAAAAAATAAGGAAAATATTAGGGATAACAGGGTAATACGGATTAG TTTTATTGGTTCATATTTATTTAATAAATAATAAAAAATAAGGAAAATATTAGGGAT ATTGATGCTAATGTTGATGATC GTTGCATTCGATTCCTGTTGTATTTAGGAGTTTAAATAAGTATGGCCAGCCATTACGCTCG TGTTATACTTACAAAAAAGCTCCTAAATATTTAGGAGTTTAAATAAGTATGGC CTTCGGAGGGCTGTCACC AATCTACTCCACCAGATAATC

GATAGAAAAGGAAATATTAAATAGGGATAACAGGGTAATACGGATTAG ACCTAAAAACATCAATATATCATATATAAGATAGAAAAGGAAATATTAAATAGG GCAACTATTAAAATTGGTGATG GTTGCATTCGATTCCTGTTGTAAATTTAATTTATTGTTTAGGCCAGCCATTACGCTCG ATTAATAAAAACTATCCATGAAGGATAGTTTAAATTTAATTTATTGTTTAGGCCAG CTTCGGAGGGCTGTCACC CAATAGTTTTAGAAGTACTTGG

AATAGAAATGAGGTGTTAATTAGGGATAACAGGGTAATACGGATTAG TATAAGATTTGATTAACTAAAAAATAATAAAATAGAAATGAGGTGTTAATTAGG TCCTGTTTCAAAATATGAAGATG GTTGCATTCGATTCCTGTTGAAGTATAAGTTTAGGAGATATTGGCCAGCCATTACGCTCG CTAATAACTAAAATTTTGTTAAGTTGTAAAGTATAAGTTTAGGAGATATTGG CTTCGGAGGGCTGTCACC AAGTTTCAATAACAAGAACTTCAG

TTCAATTTTAATCACCATTATAGGGATAACAGGGTAATACGGATTAG GAATTATTAGATAATATTTTCTTATTTTTATTCAATTTTAATCACCATTATAGGG GATTTCACGAATTTCAATTTGC GTTGCATTCGATTCCTGTTGTCTATTGAAGGAAGTGATGATAGGCCAGCCATTACGCTCG AAATAATTATACAAACATAAACAAATAATCTATTGAAGGAAGTGATGATAG CTTCGGAGGGCTGTCACC ACAAAATGCTGCTAGTGATTG

CTTGAATTAAAAATAATGACTAATAGGGATAACAGGGTAATACGGATTAG TTAAAGGAAAAATTAAGATTATAATATCTTGAATTAAAAATAATGACTAATAGG AGTGTGATGATTTAAAAGAATTTG GTTGCATTCGATTCCTGTTGGATATAAACTAATATTGCAACTTTGGCCAGCCATTACGCTCG TCTATATTATTTTATATGATTAATTAGATATAAACTAATATTGCAACTTTG CTTCGGAGGGCTGTCACC TCCACTACTTCCTGTTGAAAC

ATAGTTTTAATAGGAGAAAATTAGGGATAACAGGGTAATACGGATTAG GAAAATCAAAATATTTATAAATTAAATTAATAGTTTTAATAGGAGAAAATTAGGG GGTATGGGTAAAGGATTTTATG GTTGCATTCGATTCCTGTTGGCTAATAAATTTTAGTTATTTAGGCCAGCCATTACGCTCG GTTTTTAAAGTAATAAATTAATAAATTTGCTAATAAATTTTAGTTATTTAGGCC CTTCGGAGGGCTGTCACC CTTTAATCATATTACTCCTTTTAC

GATTTTAAGATTGGAACATATTTAGGGATAACAGGGTAATACGGATTAG TTAGTAATGTTTTGATATTATTAAAATAGATTTTAAGATTGGAACATATTTAGG AGTTCACGGATCTGATTTAAG GTTGCATTCGATTCCTGTTGGTTTTCCTCCTCTACACCTGCATTTTTTATTTAAGGCCAGCCATTACGCTCG TAATAAAAAAATGCAGGTTTTCCTCCTCTACACCTGC CTTCGGAGGGCTGTCACC TTTACACAAAACAATTGACTCAG

ATAACATCTAAAAAACTTTAGGGATAACAGGGTAATACGGATTAG TTGTTTTTTCTCCTATCTTCTAGTTTTATTATATAACATCTAAAAAACTTTAGGGATAA AGTGTTCAGCAATTCCTTGG GTTGCATTCGATTCCTGTTGATAGAAGATTAAAAAAATCGTAAGGCCAGCCATTACGCTCG AACAATAATCAATCTAAAAATCCTTTTATAGAAGATTAAAAAAATCGTAAGG CTTCGGAGGGCTGTCACC GAATCTATTAGAATAATGTTTTATG

GAAGAAAATTAGGATTAAAGCTTAGGGATAACAGGGTAATACGGATTAG TTATATAAACTATAATATAGAAAGAATAGAAGAAAATTAGGATTAAAGCTTAG CTTTTTTCATAAAACCAGACTTTC GTTGCATTCGATTCCTGTTGAGCCTTTTATTATATTAATTTTTTGGCCAGCCATTACGCTCG TAAAAACAAAGGCTTGGATAATTCCAAGCCTTTTATTATATTAATTTTTTGGC CTTCGGAGGGCTGTCACC ATGCACAAAAACCTGAAGTTG

ATACTTATATGAGGTGAGATTTAGGGATAACAGGGTAATACGGATTAG ATTTAGTTACTTTTAAATATTTTTTTCTAATACTTATATGAGGTGAGATTTAG AGAAGCTAGAAAACGTGAATAC GTTGCATTCGATTCCTGTTGTGTTAAAATAAGAAAAGGTAAATGGCCAGCCATTACGCTCG AATATCAAAATATGATATTTAATATATTGTTAAAATAAGAAAAGGTAAATGG CTTCGGAGGGCTGTCACC GTATCAACAATTATTGCAGGTC

ATACTTATATGAGGTGAGATTTAGGGATAACAGGGTAATACGGATTAG CAAAAAACTGTACTAGTCAGTTTAAATGAACCTTTAATTTAAACCAGTTATAGG GATATTTCAATATCTGGACTAG GTTGCATTCGATTCCTGTTGTGTTAAAATAAGAAAAGGTAAATGGCCAGCCATTACGCTCG TGTTTTATGTTATAAAAAAGTTAGTAACATAGATATAAAAAGGAAGAGATGGC CTTCGGAGGGCTGTCACC AATGCATGCTTATAATGAGTATG

AATATTTGTTTATACAATCTTAGGGATAACAGGGTAATACGGATTAG CTATAGTTCTATTAAAAATAAGCATTAAAAAATATTTGTTTATACAATCTTAGGGATA CATCAGCTGTTGGCTCAG GTTGCATTCGATTCCTGTTGAAAGAAAGGCAGTAGATATATGGCCAGCCATTACGCTCG TACATCTCTTATTTATTTTTATTAATAATAAAGAAAGGCAGTAGATATATGG CTTCGGAGGGCTGTCACC AATTTAAAGAACTCATATGAGTTC

GATAACTACTAAAAAGCTTTTAATAGGGATAACAGGGTAATACGGATTAG ATTACTATAACAAACTTAATAAAACAAGATAACTACTAAAAAGCTTTTAATAG GACTCGCCTATGTACGTG GTTGCATTCGATTCCTGTTGATGAACTTAAAAGTTCATTTAGGCCAGCCATTACGCTCG TATTTAACAACTATTTTTACATAAAAAAAATGAACTTAAAAGTTCATTTAGGC CTTCGGAGGGCTGTCACC GACGCATTGGACTTAAAATCC

080

39  genes  and  gene  clustersamplification  of  the  core6  cassette  (2  rounds  of  PCR) Junction  PCR

0014-­‐0016

0019-­‐0024

0035-­‐0038

041

0050-­‐0060

0072-­‐0075

0077-­‐0078

0272-­‐0277

0083-­‐0093

0204-­‐0212

0217-­‐0219

0223-­‐0226

0236-­‐0237

0231-­‐0232

0241-­‐0246

0251-­‐0252

0255-­‐0256

0261

0268-­‐00269

0454-­‐0474

0292-­‐0293

0331-­‐0332

0349

0354

0357-­‐0358

0383-­‐0386

0393

0395-­‐0397

0417

0436

0444

0476-­‐0477

0480

0483-­‐0486

0489-­‐0492

0494-­‐0498

0503-­‐0505

0367-­‐0369

   

73    

MPCR  set   Sequence  

Amplicons  (bp)  

Set  9  (WT)  

CTACTTGTTCACCTTCTCTG  121  

GTGATATAGATGATACAATAAAGA  GTAGAACATGTTGCAGATAAG  

204  TGATGGTGCATAACGTAATC  ATTTAGTTGCTGATTGAGTTTG  

256  CTAGGACCAGTGGCTGC  AGTTGACTGAAATTCTCAGTC  

301  AAGATCGTTTAGCTAAAGGAC  TGGTTATATAATACTTGGAGTG  

408  GCAGGTTTTCCTCCTCTAC  CATGATTGATATGTTTAAAGATGC  

515  TGCAAAGACTAAAGTATATGTG  TTGATGTATCTCACTTATTTAGA  

612  CAACAACTAGAGATTTAAACAG  GCTTGTTCAACATTATAAGCTC  

724  TTGATGGTGACTCTAATAATACT  

Set  9  (RGD)  

ATTCCGGACTAAAATCCAGG  129  

GTGATATAGATGATACAATAAAGA  TTTTAGTATGTTTAAGGAGAAGA  

186  TGATGGTGCATAACGTAATC  CATATGTTGCAGTTTGTGATAC  

257  GGAGAAAACTAAATAACTGTTC  TCATGTTAAATACTGCTTGTCA  

306  AAGATCGTTTAGCTAAAGGAC  GATACAAATGATCAAATAGAAGC  

400  GCAGGTTTTCCTCCTCTAC  GTTTTACCAACTCCAGGTTC  

486  TGCAAAGACTAAAGTATATGTG  GTGAAGTTAATATTTTTGAACCA  

618  CAACAACTAGAGATTTAAACAG  ATCTTGTTATCTTCCAGCAG  

724  CTCTATCTGGATCTGAAGCT  

Set  10  

AAAGTAGTGTTATGTGGAGC  108  

ATATAGAAACTATGATGAACAC  TGACTGTTAGTTTACTATCAG  

176  AGAGTTAAGTTTTAATACACTTG  CACGACTACCTTCTTCTAAG  

266  TATGTCAAAAAGACAAGCTTTAG  CAAGTTGAACGTGGAGCT  

323  ACACTATTAGCAGGAGCTG  GAAGTTGGTGGTTTAACTC  

412  AGTTTTGATCAACTAGTTGTC  

   

74    

Table S15. MPCR primer sets used for confirming assembled genomes. Combinatorial genome assembly was performed using syn1.0 and RGD segments. Each MPCR primer set contains 8 primer pairs to produce amplicons representing each 1/8th segment (syn1.0 or RGD). Set 9 (syn1.0) produces 8 amplicons only from syn1.0 segments and Set 9(RGD) only produces 8 amplicons from RGD1.0 segments. Set 10 primer mix can produce 8 amplicons from both syn1.0 and RGD segments. Similarly, Set 15 and 16 can specifically detect syn1.0 and RGD2.0 segments, respectively in assembly 3 and 4 (see Table S7).

CAGCATCTCCTGTAAAGTG  513  

GATTGTTTGTAACTGAAGCTAG  CTAGTTTCTTCTTCACTAAATG  

619  CAACTGTTGGAGATGTTGC  CCACGTGATCCTCTTAAAG  

741  AATACACCATTTGAAGGTGG  

Set  15  

ATTCCGGACTAAAATCCAGG  129  

GTGATATAGATGATACAATAAAGA  TTTTAGTATGTTTAAGGAGAAGA  

186  TGATGGTGCATAACGTAATC  CATTATAATAACGATGAATTCTAG  

268  GAATTTCACGAGCGAAAATTC  GAAGTAGTTCCTGAAGAGATTC  

314  TCACCATCTTTTGAAGTATAAGC  GATACAAATGATCAAATAGAAGC  

400  GCAGGTTTTCCTCCTCTAC  GTTTTACCAACTCCAGGTTC  

486  TGCAAAGACTAAAGTATATGTG  GTGAAGTTAATATTTTTGAACCA  

618  CAACAACTAGAGATTTAAACAG  ATCTTGTTATCTTCCAGCAG  

724  CTCTATCTGGATCTGAAGCT  

Set  16  

CTACTTGTTCACCTTCTCTG  129  

GTGATATAGATGATACAATAAAGA  ACAATTGGTGCTGGAACAG  

220  CATGTAAGTTACTCTCTTTAGC  ATTTAGTTGCTGATTGAGTTTG  

258  CTAGGACCAGTGGCTGC  AGTTGACTGAAATTCTCAGTC  

301  AAGATCGTTTAGCTAAAGGAC  TGGTTATATAATACTTGGAGTG  

408  GCAGGTTTTCCTCCTCTAC  CATGATTGATATGTTTAAAGATGC  

515  TGCAAAGACTAAAGTATATGTG  TTGATGTATCTCACTTATTTAGA  

612  CAACAACTAGAGATTTAAACAG  TCTACAGTTTTCATATACGCC  

718  GTATTGGTTTTAACTTATGATGA  

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