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NotebookComposite parts assembly

Wednesday 15/08/18 Level 1 Golden Braid reaction for BBa_K2656105

plasmid ID Promoter RBS CDS terminator

BBa_K2656105

BBa_K2656004

BBa_K2656009

BBa_K26560222

BBa_K2656026

Tuesday 21/08/18 Adri Level 1 Golden Braid reaction for: BBa_K2656105 with 50 ng linearized destination vector


BBa_K2656111

BBa_K2656004

BBa_K2656009

BBa_K2656020

BBa_K2656026

BBa_K2656112

BBa_K2656004

BBa_K2656009

BBa_K2656021

BBa_K2656026

Alberto: BBa_K2656109 (BBa_K2656014) BBa_K2656113 (BBa_K2656018) BBa_K2656125(BBa_K26560142) BBa_K2656110 (BBa_K2656024) BBa_K2656126(BBa_K2656025) they all share the promoter (BBa_K2656004), rbs (BBa_K2656009), and terminator (BBa_K2656026) Wednesday 22/08/18 Adri Transformation results: There are yellow colonies in the plates with BBa_K2656111 and with BBa_K2656112 Testing the functionality of BBa_I79005 GB domesticated with different RBS Level 1 Golden Braid reaction for:


BBa_K26561093

BBa_K2656000

BBa_K2656008

BBa_K2656022

BBa_K2656026

BBa_K26561094

BBa_K2656000

BBa_K2656009

BBa_K2656022

BBa_K2656026

BBa_K26561095

BBa_K2656000

BBa_K2656010

BBa_K2656022

BBa_K2656026

BBa_K26561096

BBa_K2656000

BBa_K2656011

BBa_K2656022

BBa_K2656026

BBa_K26561097

BBa_K2656000

BBa_K2656012

BBa_K2656022

BBa_K2656026

Transformation of pITU 6,7,8,13,14,15,16,17 by electroporation Berto: Testing the funcionality of different promoters by using the previous working parts and only changing the promoter:

plasmid promoter rbs cds terminator

BBa_K26 pACA5 BBa_K26 BBa_K26 BBa_K265

561098 56009 56022 6026

BBa_K26561099

BBa_K2656002

BBa_K2656009

BBa_K2656022

BBa_K2656026

BBa_K2656106

BBa_K2656005

BBa_K2656009

BBa_K2656022

BBa_K2656026

BBa_K2656107

BBa_K2656007

BBa_K2656009

BBa_K2656022

BBa_K2656026

Carol Level 1 Golden Braid reaction for:


BBa_K2656101

BBa_K2656004

BBa_K2656009

BBa_K2656013

BBa_K2656026

BBa_K2656108

BBa_K2656004

BBa_K2656009

BBa_K2656023

BBa_K2656026

BBa_K26561091

BBa_K2656004

BBa_K2656009

BBa_K2656016

BBa_K2656026

BBa_K26561092

BBa_K2656004

BBa_K2656009

BBa_K2656017

BBa_K2656026

Transformation of 10G electrocompetent cells with 2 uL of the GB reaction ligation products. Plating in Km+Chromomax LB-LB-agar (50 and 300 uL per plate). Thursday 23/08/18 Adri: Results of the transformation:

● BBa_K2656111 and 8 are yellow -> YFP is correct ● BBa_K2656105 is green -> we can use a linearized destination plasmid. But there is a

lot of background. I'm doing a cPCR to know what is that background. ● BBa_K26561093-17 are not green --> we will do a cPCR to know what is growing

cPCR results: 1: ladder 2: BBa_K26561094 blue colony 1 3: BBa_K26561094 blue colony 2, 4: BBa_K26561094 white colony 1, 5: BBa_K26561094 white colony 2 6: C+ 7: BBa_K2656105 green colony 1 8: BBa_K2656105 green colony 2 9: BBa_K2656105 green colony 3, 10: BBa_K2656105 white colony 1, BBa_K2656114: BBa_K2656105 white colony 2, 12: BBa_K2656105 white colony 3 13: C-, 14: ladder

BBa_K26561094 white colony 1 may be correct. White colonies of BBa_K2656105 with linearized destination plasmid are the result of a recircularization of the destination vector. I will use less destination plasmid in the next assembly. Carol BBa_K2656105 and BBa_K2656108 are correctly assembled (sfGFP and GFP+LVA tag as reporter): they express fluorescence. Inoculate 3 colonies of each to make stock. cPCR of 6 colonies for BBa_K26561091 (luxR) and BBa_K26561092 (AraC). Annealing time: 1 min. Run electrophoresis gel (1%) ladder, colonies 1-6 of BBa_K26561091, colonies 1-5 of BBa_K26561092, control+, ladder, colony 6 of BBa_K26561092, ladder

Colony 1 for pITUo11 (luxR) has the correct length. Inoculare 5 uL to do stock. Any colony for BBa_K26561092 has the TU, so I pick 7 more colonies to do cPCR. ladder, colonies 1 to 7, C+ (pGreen alpha1), ladder

The length of the fragments are not around 1000 pb (AraC colony PCR amplicon). Berto: picked and grew 3 colonies from TU1 and TU4, which were expressing the rfp. Colony pcr of 5 colonies for TUs 2, 3 and 5 Transformed yesterday's GG reaction of BBa_K26561098,19,20,21 and plated. We'll see what happens tomorrow Friday 24/08/18 Berto: Yesterday I transformed pITUs 18,19,20,21 and they are correct. Promoters (pACA 5, 12, 17 and 20) are correct There are fluorescent colonies in all the plates Results for yesterday's cPCR: ladder, BBa_K2656113 (1, 2, 3, 4, 5), BBa_K2656125(1,2,3,4,5), positive control, negative control, ladder ladder, BBa_K2656126(1,2,3,4,5), positive control, negative control, empty wells, ladder

pITU 2 and pITU 5 didn´t work properly, so pACA 18 and pACA 33 (blue chromoprotein and mint smell) have not been correctly assembled. Repeat minipreps pITU 3 has been correctly assembled, so we need to know if pACA 22 (luxI) is functional or not Monday 27/08/18 Adri: assembly of BBa_K2656105 with different amounts of linearized destination plasmid (35, 30, 25, 20, 15 and 10 ng) Transformation by electroporation: the reaction with 10 ng of destination plasmid is not being transformated and the reaction with 15 ng of destination plasmid has problems too. Carol: RBS functionality testing using sfGFP as reporter.


BBa_K2656100

BBa_K2656004

BBa_K2656008 (J61100)

BBa_K2656013

BBa_K2656026

BBa_K2656102

BBa_K2656004

BBa_K2656010 (B0032)

BBa_K2656013

BBa_K2656026

BBa_K2656103

BBa_K2656004

BBa_K2656011 (B0034)

BBa_K2656013

BBa_K2656026

BBa_K2656104

BBa_K2656004

BBa_K2656012 (J61101)

BBa_K2656013

BBa_K2656026

Transformation of 10G electrocompetent cells with 2 uL of the GB reaction ligation products. Plating in Km+Chromomax LB-agar (50 and 300 uL per plate). Tuesday 28/08/18

Adri Transformation results: The lowest background was obtained from the reaction with 25 ng of linearized destination plasmid

Carol BBa_K2656103: colonies express the sfGFP. Pick 3 of them to do stock. BBa_K2656100, 23 and 25 are TUs with weak promoters, so I am going to do cPCR of 6 colonies for each TU in order to corroborate they are expressing the GFP. Ann. time: 1 min 10 sec. Run electrophoresis gel (1%) ladder, c. 1 to 5 BBa_K2656100, c. 1 to 5 BBa_K2656102, C+, C- ladder ladder, c. 1 to 5 BBa_K2656104, C+, ladder

To do stock: inoculate colonies 2, 3 and 5 for BBa_K2656100, colonies 1, 3 and 5 for BBa_K2656102, colonies 1, 2 and 3 for BBa_K2656104 in LB+Km. Berto: Repeat the GB assembly BBa_K2656113 (BBa_K2656018) BBa_K2656126(BBa_K2656025) By using the new minipreps of both colonies in the stock Thursday 30/08/18 Carol Testing the functionality of the RBS pieces without the BB scar: Golden Braid Level 1 assembly for the AraC gene repeated again) with new Miniprep of colony 1 stock (pITU30)


BBa_K26561136

BBa_K2656004

B0032 BBa_K2656013

BBa_K2656026

BBa_K26561137

BBa_K2656004

J61101 BBa_K2656013

BBa_K2656026

BBa_K26561138

BBa_K2656004

B0030 BBa_K2656013

BBa_K2656026

BBa_K26561139

BBa_K2656004

B0034 BBa_K2656013

BBa_K2656026

pITU30 BBa_K2656004

J61100 BBa_K2656013

BBa_K2656026

pITU31 BBa_K2656004

BBa_K2656009 BBa_K2656013

BBa_K2656026

Adri: Testing T7 phague promoter with B0032 and B0034 in pGreen alpha 1 and pLX-B2 alpha 1

BBa_K26561095

BBa_K2656000

BBa_K2656010

BBa_K2656022

BBa_K2656026

BBa_K26561096

BBa_K2656000

BBa_K2656011

BBa_K2656022

BBa_K2656026

BBa_K2656123

BBa_K2656000

BBa_K2656010

BBa_K2656022

BBa_K2656026

BBa_K2656124

BBa_K2656000

BBa_K2656011

BBa_K2656022

BBa_K2656026

Berto: Transformed new BBa_K2656113.1, 2.2, 5.1 and 5.2 Colony PCR of 5 colonies of each pITU 20 and 21 and 2 cultures of 2 colonies from pITU 21 which were the only that grew electroforesis results: ladder, BBa_K2656106(1-5), c+, c-, empty wells, ladder ladder, BBa_K2656107(1-5), colony day 27, colony day 28, c+, c-, empty wells, ladder

Friday 31/08/18 Transformation of 10G electrocompetent cells with 2 uL of the GB reaction ligation products. Plating in Km+Chromomax LB-agar (50 and 300 uL per plate). BERTO: Pitu 2.2 IS FINALLY BLUE!!!! Cpcr of Pitu 5.1 AND 5.2: Electrophoresis results: ladder, pITU 5.1(1-10), c+,c-, ladder ladder, pITU 5.2(1-10), c+,c-, ladder

Saturday 1/09/18 cPCR for pITU31 (transcriptional unit with AraC). Ext time: 1 min

Electrophoresis gel (1%) result: any colony carried the TU. I will repeat the GB reaction with AraC the colony 2 from stock. Monday 3/09/18 cPCR of BBa_K26561136 to 30 (RBS without BB scar). Ann. time: 1 min. Electrophoresis gel (1%): ladder, colonies 1-4 BBa_K26561136, colonies 1-4 BBa_K26561137, ladder

ladder, colonies 1-4 BBa_K26561138, colonies 1-4 BBa_K26561139, colonies 1-3 pITU30, C+, ladder

Colonies for BBa_K26561136 and 27 carry the TUs (1000 pb amplicon). Inoculate colonies 1, 3 and 4 of BBa_K26561136 and c. 1, 2, 3 BBa_K26561137 to do stock. No correct assembly for BBa_K26561138, 29, 30. From stock: inoculate colony 2 carrying AraC gene into LB+Km liquid medium to do Miniprep. Adri: T7 promoter test results: Colonies transformed with pLX and the construction have expression. Colonies transformed with pGreen and the construction have not expression. We can not use the T7 promoter with a high copy number destination vector. GB assembly of:


pITX3 BBa_K2656004

BBa_K2656010

BBa_K2656022

BBa_K2656026

pITX4 BBa_K2656004

BBa_K2656011

BBa_K2656022

BBa_K2656026


pITU34 BBa_K2656004

BBa_K2656010

BBa_K2656022

BBa_K2656026

BBa_K2656117

BBa_K2656004

BBa_K2656011

BBa_K2656022

BBa_K2656026

Tuesday 4/9/2018 Adri Transformation by electroporation of pITx 3 and 4 and pITU 34 and 35 Wednesday 5/9/2018 Carol Miniprep of AraC domesticated piece from the stock colony 2. DNA conc: 37.5 ng/uL. With this DNA piece, GB assembly of BBa_K26561092. GB assembly of pITU36, 37, 38 and 39


pITU36 BBa_K2656003

BBa_K2656011

BBa_K2656022

BBa_K2656026

pITU37 BBa_K2656003

BBa_K2656011

BBa_K2656023

BBa_K2656026

pITU38 BBa_K2656003

BBa_K2656011

BBa_K26560142

BBa_K2656026

pITU39 BBa_K2656003

BBa_K2656011

BBa_K2656015

BBa_K2656026

Transformation in 10G electrocompetent cells with 2 uL GB. Plating in Km medium. Adri: Transformation results: -pITX3 has not colonies -pITX4 has expression -pITU34 has not expression -BBa_K2656117 has expression Thursday 6/09/2018 Carol: cPCR of BBa_K26561092 and pITU36-39 Colony 1-6 for each pITU. ladder, BBa_K26561092 (1-6), pITU36 (1-6), ladder, pITU37 (1-6), pITU38 (1-6), C+, ladder

ladder, pITU39 colony 1 to 6, C-, ladder

Any colony has the desired TU. Monday 10/09/18 GB assembly of BBa_K26561100 to BBa_K26561105:


BBa_K26561100

BBa_K2656004

BBa_K2656010

BBa_K2656014

BBa_K2656026

BBa_K26561101

BBa_K2656004

BBa_K2656011

BBa_K2656014

BBa_K2656026

BBa_K26561102

BBa_K2656004

BBa_K2656010

BBa_K2656024

BBa_K2656026

BBa_K26561103

BBa_K2656004

BBa_K2656011

BBa_K2656024

BBa_K2656026

BBa_K26561104

BBa_K2656004

BBa_K2656010

BBa_K2656018

BBa_K2656026

BBa_K26561105

BBa_K2656004

BBa_K2656011

BBa_K2656018

BBa_K2656026

10G electrocompetent transformation. Plating in Km solid medium. Incubate overnight.

Tuesday 11/09/18 BBa_K26561100 is correctly assembled (there are red colonies). Pick 3 colonies to do stock. BBa_K26561105 is correctly assembled (there are blue colonies). Pick 3 colonies to do stock. cPCR BBa_K26561092 (6 colonies), 32 (1 colony), 33 (4 colonies), BBa_K2656118 (6 colonies), 42 (6 colonies), 43 (6 colonies), 44 (5 colonies), C+, C-. After running electrophoresis gel (1%), only BBa_K26561100 colonies 1, 4 and 5 have the correct amplicon size. Pick them to do stock. Friday 14/09/18 Adri Assembly of BBa_K2656105 with: Circular destination plasmid Digested with BsaI destination plasmid Digested with BsaI and SAP destination plasmid The thermocycler broke during the reaction Monday 1/10/18 GB assembly of BBa_K26561106, BBa_K2656121 (using BBa_K2656017 from 2 colonies) and BBa_K26561108


BBa_K26561106

BBa_K2656006

BBa_K2656011

BBa_K2656022

BBa_K2656026

BBa_K26561107.1

BBa_K2656004

BBa_K2656011

BBa_K2656017.1

BBa_K2656026

BBa_K26561107.2

BBa_K2656004

BBa_K2656011

BBa_K2656017.2

BBa_K2656026

BBa_K26561108

BBa_K2656003

BBa_K2656011

BBa_K2656022

BBa_K2656026

10G electrocompetent transformation. Plating in Km solid medium. Incubate overnight. Tuesday 2/10/18 Pick 8 colonies of each plate for BBa_K26561106, BBa_K26561107 and BBa_K26561108 to do Miniprep Wednesday 3/10/18 Miniprep. DNA conc:

Name A260/A280 DNA conc. (ng/uL)

46.1 1,931 237,188 46.2 1,906 157,903 46.3 1,915 210,545 46.4 1,918 191,449 46.5 1,895 227,438 46.6 1,904 288,567 46.7 1,931 214,405 46.8 1,893 60,061


BBa_K2656121.1.1

1,974 57,133

BBa_K2656121.1.2

1,897 88,519

BBa_K2656121.1.3

1,894 43,218

BBa_K2656121.1.4

1,852 27,8

BBa_K2656121.1.5

1,927 141,133

BBa_K2656121.1.6

1,892 60,795

BBa_K2656121.1.7

1,886 73,596

BBa_K2656121.1.8

1,887 69,867


BBa_K2656121.2.7

1,917 41,057

BBa_K2656121.2.8

2 5,666

BBa_K2656121.2.1

1,904 33,465

BBa_K2656121.2.2

2,15 14,072

BBa_K2656121.2.3

2,045 22,463

BBa_K2656121.2.4

2,123 18,646

BBa_K2656121.2.5

2,803 37,019

BBa_K2656121.2.6

2,019 41,871


BBa_K2656122.1

1,905 158,142

BBa_K2656122.2

1,856 265,557

BBa_K2656122.3

1,879 133,416

BBa_K2656122.4

2,054 31,933

BBa_K2656122.5

2,14 86,65

BBa_K2656122.6

1,903 196,811

BBa_K2656122.7

1,903 141,644

BBa_K2656122.8

1,892 134,291

Friday 5/10/18 Carol GB assembly of BBa_K26561109 and pITU50:


BBa_K26561109

BBa_K2656005 BBa_K2656009

BBa_K2656022

BBa_K2656026

pITU50 BBa_K2656007 BBa_K2656009

BBa_K2656022

BBa_K2656026

Assembly of BBa_K2656105 with circular destination plasmid digested with BsaI and SAP. 10G electrocompetent transformation. Plating in Km solid medium. Incubate overnight. Digestion of BBa_K26561106, BBa_K2656121, BBa_K2656122 with BsmBI endonuclease. Run electrophoresis gel (1%). 1: ladder, 2-6: BBa_K26561106, 7-9: BBa_K26561107, 10: ladder; 1: ladder, 2: BBa_K26561106, 3-6: BBa_K26561108, ladder

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