igem 2004 review
DESCRIPTION
iGem 2004 review. S ignificant differences between initial and final design. 0. 0. 1. 0. 0. 0. 0. 1. 1. 0. Int 1. Xis 1. Int 2. Xis 3. Int 2. Xis 2. Initial design. PLtetO. rbs. xis2. attB. rbs. gfp. attP*. t0. rbs. int2*. Final design. - PowerPoint PPT PresentationTRANSCRIPT
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