iGem 2004 review

Significant differences between initial and final design.

Initial design

Final design

xis2 attB rbs gfp attP*rbsPLtetO

rbs int2*

t0

Int1

0 0

Xis1 Int2 Xis2 Int2 Xis3

1 100

0

00

1

How did this work, and what was the problem?

Int1

0 0

Xis1 Int2 Xis2 Int2 Xis3

1 100

0

00

1

• Counting mechanism:– Initial state: 0 0 0– Pulse 1: 1 0 0– Pulse 2: 0 1 0– etc. . . .

• Race condition problems between each Int and Xis:Ordering of signal arrival for an input is critical for correct

behaviorPossible erroneous outputs caused by latency?

Design 1. Slide 59

First design: two half-(?) bits that are coupled.

AttR Term AttL*Int 2 X 2 GFP

AttP Term AttB*Int 1 X 1 CFP

Pulse 1

Pulse 2

Design 2. Slide 9: pulse 1a:0,2a:YFP,1b: GFP,2b:0

Two bits

AttR Term AttL*Int 2 X 2 GFP

AttP Term AttB*Int 1 X 1 CFP

Pulse 1

Pulse 2

Pulse 1aOutput : 0 (state 1)

AttR Term AttL*Int 2 X 2 GFP

AttR

Term

AttL*Int 1 X 1 CFP

Pulse 1

Pulse 2

Pulse 2aOutput : Yellow (state 2)

AttP

Term

AttB*Int 2 X 2

AttR

Term

AttL*Int 1 X 1 CFP

Pulse 1

Pulse 2

GFP

Pulse 1bOutput : Green (state 3)

AttP

Term

AttB*Int 2 X 2

AttP Term AttB*Int 1 X 1

GFP

Pulse 1

Pulse 2

CFP

Pulse 2bOutput : No (state 1)

AttR Term AttL*Int 2 X 2

AttP Term AttB*Int 1 X 1 CFP

Pulse 2

Pulse 1

GFP

Blue Heron design differs slightly. Why?

AttR Term AttL*Int 2 X 2 GFP

AttP Term AttB*Int 1 X 1 CFP

Pulse 1

Pulse 2

Design 2. Slide 9: pulse 1a:0,2a:YFP,1b: GFP,2b:0

Design 3. Slide 11: 1a:0, 2a: 0, 1b: YFP, 2b: GFP

P22 Xis +AAV

EYFP +AAV

p22 Int+ LVA

BBa_E0034 BBa_I11030 BBa_I11031

λ attP

BBa_I11023

Terminator

BBa_B0013

λ attB (rev comp,

2)BBa_I11022 BBa_I11061 :

p22 Half Bit

λ Xis +AAV

ECFP +AAV

λ Int+ LVA

BBa_E0024 BBa_I11020 BBa_I11021

p22 attP

BBa_I11033

Reverse Terminator

BBa_B0025

p22 attB (rev comp)

BBa_I11032

λ Half BitBBa_I11060 :

These[1] were synthesized, all now Bio-bricks. However, they were not completed by the time of

the presentation. Work shown in the following slides indicates that this design will not work.

P22 Xis +AAV

EYFP +AAV

p22 Int+ LVA

BBa_E0034 BBa_I11030 BBa_I11031

λ attP

BBa_I11023

Terminator

BBa_B0013

λ attB (rev comp,

2)BBa_I11022 BBa_I11061 :

p22 Half Bit

[1] Differ slightly from design as described. Pulse 1a: P22 expressed, no signal, flip bit 2 to make terminator and L, R sites. Pulse 2a: alpha intergrase expressed, no signal, flip bit 1 to make no terminator and L, R sites. Pulse 1b: express p22 int and xis, yfp, flip bit 2 to make no terminator and P, B sites. Pulse 2b: express alpha int and xis, GFP, flip 1 to make terminator and P, B (back to initial state).

[2] Means B*?

λ Xis +AAV

ECFP +AAV

λ Int+ LVA

BBa_E0024 BBa_I11020 BBa_I11021

p22 attP

BBa_I11033

Reverse Terminator

BBa_B0025

p22 attB (rev comp)

BBa_I11032

λ Half BitBBa_I11060 :

For testing, why was reporter between flip sites?

GFPAttP AttB*

Design 4 / Test . Slide 13: Turn green when terminator in reverse position?

Design 3. Doesn’t work. 1. Can’t read through attP. 2. Cloning problem in Int construct. 3. Overlaps (between attP & end of Int, and beginning of Int & end of Xis).

Int XisIPTG Ara

GFPAttP AttB*

Int XisIPTG Ara

Construct to test inversion“Description has that system will green when terminator is in

the reverse position,” though this not clearly depicted.

Xis

Int

attP

attB*

origin

Kan

T0

GFP_AAV

PLlacO PLtetO

ECFP +AAV

p22 attPReverse

Terminatorp22 attB

(rev comp)

Inverting lambda and GFP? Why?

Not designed?

Failure analysisOverlap implies cross talk between Int and Xis or

binding of wrong region of Int / Xis to site?

Xis

Int

PLlacO PLtetO

GFP_AAV

attP

attB*

origin

Kan

dh5aZ1

Can’t read through attP

Beginning of Int andend of Xis overlap by 40 amino acids [1]

End of Int and attPOverlap [2]

Can’t continue after KanR

Cloning problem near

PLlacO in lambda

construct (SalI) T0

[1] Cross talk? and [2] Non-specific binding?

Failure analysisSeems that one clear problem with reading through att

site

GFP_AAV

attP

attB*

PLtetO

GFP_AAV

PLtetO

No GFP GFP

First two designs shown are pretty similar. Reasons for difference not clear.

For test, extrapolate that 2/3 won’t work : can’t have AttP before reporterLots of additional points:1. Reverse AttP and B sites. 2. Mutagenize erroneous AttP site on int to eliminate overlaps?3. Question : is there enough int? What?4. How to measure levels of xis and int? Why?5. Int binding block read-through?6. Need a new strain? Associated between E. Coli genome attB and construct

P site?7. Consider Gateway system (design 5 informed by this)8. AttB sites can be read through only if RBS is after AttB1

AttR Term AttL*Int 2 X 2 GFP

AttP Term AttB*Int 1 X 1 CFP

Pulse 1

Pulse 2

Possible new design

PLlacO

Lambda Int

p22 attP

p22 attB*

Lambda Xis

GFP_AAV

pSC101

Kan

p22 Xis

Lambda attB*

Lambda attP

p22 Int

PLtetR

Switch so that it reads throughB* site, rather than attP?

Again, why inverting full lambda and GFP?

Concerns remained

PLlacO

Lambda Int

p22 attP

p22 attB*

Lambda Xis

GFP_AAV

pSC101

Kan

p22 Xis

Lambda attB*

Lambda attP

p22 Int

PLtetR

Enough integrase? What do they mean by enough?

How to measure levels?Why do they need to?

Int binding blocks read-thru?

Again, why inverting full lambda and GFP?

Need for a new strain?attP integration into host chromosome?

So, looked into designs used by the Gateway system

Gateway [1] uses three methodsPromoter – attB1 – rbs – gene of interest – attB2Promoter – rbs – Fusion – attB1 – gene of interest – attB2Promoter – attB1 – rbs – gene of interest – attB2 – Fusion

[1] http://www.bioresearchonline.com/article.mvc/GATEWAY-Cloning-TechnologyA-Universal-Cloning-0001

With this in mind, design shifted slightly.

Gateway [1] uses three methodsPromoter – attB1 – rbs – gene of interest – attB2Promoter – rbs – Fusion – attB1 – gene of interest – attB2Promoter – attB1 – rbs – gene of interest – attB2 – Fusion

PLlacO Lambda Int

p22 attP

p22 attB*

Lambda Xis

GFP_AAV

pSC101Kan

p22 Xis

Lambda attB*

Lambda attP

p22 Int PLtetR

Xis-attB-GFP junction. want to make a protein across the junction

GFP-attP-terminator We want the attP and a transcriptional terminator to follow the GFP

First two designs shown are pretty similar. Reasons for difference not clear.

Design 4: Xis-attB-GFP junction (make a protein across the junction) and GFP-attP-terminator

TermGFP AttPAttB*Int 1 X 1

xis attB rbs gfp attP*rbsPLtetO

rbs int*

t0

Design 5: Put int in same operon as GFPWhat was done with overlaps?Is there enough int?Was this built (what about the Blue Heron constructs)?Int binding read-through?What is the right strain?*int 58 aa coding region to allow GFP in same operon; why?

P22: xis, attB, gfp junction

xis attB rbs gfp attP*rbsPLtetO

rbs int*

F--T--M--S--*--*-- M—R—K—G- --H--D--K--L--I--T--Q--R--I--R--N--A--K--V--V--K--E--A--A--Y--A--*--

ttcatgacaagctaataacgcagcgcattcgtaatgcgaaggtcgttaaggaggcagcctatgcgtaaggaattB rbs

t0

P22: gfp-attP junction

xis attB rbs gfp attP*rbsPLtetO

rbs int*

t0

A--*--*-- taataatttttggtacttctgtcccaaatatgtcccacagtaaaaataaggaaggcacgaataatacgt\Aagtatttgatttaactggtgccgataataggagacgaacctacgaccttcgcattacgaattataagaact\accttttaagtcaacaacataccacgtcatacctgcgctcacacgtcccatcttcgaaagacatgcaaagcc\ttgcaaaccgatgcaaagatttgtatgtcccatttttgtcccaaaccacttagTerminatorggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacg\ctctcctgagtaggacaaatccgcc

Lamba bit: xis, attB, gfp junction

l xis l attB1 gfp l attP1’rbsPLtetO

rbs int*

K--A--K--S--*--*-- M—R—K—G- -R--R--S--H—N—N—K—F—V—Q—K—S—R—L—R—R—Q—A--Y—A--*

AAGGCGAAGTCAtaataACAAGTTTGTACAAAAAAGCAGGCTaaggaggcaggcctatgcgtaaggaattB1 rbs

t0rbs

Lambda: gfp-attP junction

A--*--*-- taataacatagtgactggatatgttgtgttttacagtattatgtagtctgttttttatgcaaaatctaatt\Taatatattgatatttatatcattttacgtttctcgttca(gcttttttgtacaaacttg)gcattataaaaaa\gcattgctcatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttTerminatorggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgct\ctcctgagtaggacaaatccgcc

l xis l attB1 gfp l attP1’rbsPLtetO

rbs int*

t0rbs