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