Week 2: May 23-27
From 2011.igem.org
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(→Grow up for Tomorrow's miniprep) |
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All above ligations | All above ligations | ||
Plated on AMP | Plated on AMP | ||
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#B0034+Mcherry+B0015 (kan) colony A | #B0034+Mcherry+B0015 (kan) colony A | ||
#J23113+B0034+C0051 (Amp) colony A | #J23113+B0034+C0051 (Amp) colony A | ||
#J23105+B0034+C0051 (Amp) colony A | #J23105+B0034+C0051 (Amp) colony A | ||
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===Thursday=== | ===Thursday=== |
Revision as of 13:19, 6 June 2011
Contents |
Monday
Reporters
The reporters continued research on the linkers to hold the fusion proteins.
Sensors
The sensor group ran PCR for four promoters (J23113, J23105, J23118, J23100). These constitutive promoters will exist in front of the RecA coding sequence.
All
The entire team met with Dr. Richard today to present our project. After hearing our proposal, Dr. Richard pointed out potential roadblocks and needs for fallback ideas:
For the RecA mutations, if we cannot eliminate the recombinase behavior, we may have to eliminate homologous material from our plasmid. This group should be finished in two weeks, and its members will be assigned a new task.
For reporters, in order to test the reporter, we need to use or develop an inducible promoter that will allow the reporter to emit pigment without the lambda sensor.
We also need to construct a control bar that will show if the cells in the dosimeter are dead and the device is working properly. This cell colony will feature a reporter with an inducible promoter that will be triggered by an unknown stimulus.
Dr. Richard also proposed a modeling endeavor comparing the reporting rates of Imperial’s system vs. our proposed redundant system, which features a second GFP fused to the enzyme with a linker cleaved by RecA in addition to the linker cleaved by tev.
Tuesday
Reporters
The reporter group decided on a few linkers to test in a fusion protein construct. These linkers are K243004 (4 amino acids), K105102 (10 amino acids) as well as the linker used in the Imperial College part, K316007. The reporters spent the rest of the time struggling to design primers for PCR. We needed primers for the XylE part of K316007, the linker used by Imperial College, K243004, K105012, the CI repressor/RecA cleavage site, the tev cleavage site, the GFP and flag tag complex and the LacZ part (J33210).
Primers
Note:
Green font indicates junk DNA ends (binding sites).
Red font indicates annealing sites.
CAPITAL LETTERS INDICATE PREFIX AND SUFFIX.
lowercase letters indicate gene of interest.
Linker used in K316007
- designed by Ben
Annealing Temperature: 54.13°C
Sequence:
5’ TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggaggttcaggaggcagcACCGGTTAATACTAGTAGCGGCCGCTGCAGTATT 3’
Forward:
5’ TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggaggttcagg 3'
Reverse:
5’ AATACTGCAGCGGCCGCTACTAGTATTAACCGGTgctgcctcctgaacctccGC 3’
K105102: 10 Amino Acid Linker
- designed by Brian
Annealing Temperature: 56.3°C
Sequence:
5' TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggtgaaaatttgtattttcaatctggtggtACCGGTTAATACTAGTAGCGGCCGCTGCAGTATT 3'
Forward:
5' TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggtgaaaatttgtattttcaa 3'
Reverse:
5' AATACTGCAGCGGCCGCTACTAGTATTAACCGGTaccaccagattgaaaatacaaattttcacc 3'
K243004: 4 Amino Acid Linker
- designed by Alex
Annealing Temperature: 57.78°C
Sequence:
5' TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggtggttctggtACCGGTTAATACTAGTAGCGGCCGCTGCAGTATT 3'
Forward:
5' TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggtggttctggtACC
Reverse:
5' AATACTGCAGCGGCCGCTACTAGTATTAACCGGTaccagaaccaccG 3'
tev Cleave Site
- designed by Jim
Annealing Temperature: 55.83°C
Sequence:
5' ATTAGAATTCGCGGCCGCTTCTAGATGGCCGGCgagaatttgtattttcagggtACCGGTTAATACTAGTAGCGGCCGCTGCAGATTA 3’
Forward:
5' ATTAGAATTCGCGGCCGCTTCTAGATGGCCGGCgagaatttgtattttcaggg 3'
Reverse:
5' TAATTAATCTGCAGCGGCCGCTACTAGTATTAACCGGTaccctgaaaatacaaattctc 3'
cI Cleave Site
- designed by Jim
Annealing Temperature: 56.13°C
Sequence:
5' ATTAGAATTCGCGGCCGCTTCTAGATGGCCGGCgttcaggcagggatgttctcaACCGGTTAATACTAGTAGCGGCCGCTGCAGATTA 3’
Forward:
5' ATTAGAATTCGCGGCCGCTTCTAGATGGCCGGCgttcaggcagggatgttc
Reverse:
5’ TAATCTGCAGCGGCCGCTACTAGTATTAACCGGTtgagaacatccctgcctg 3’
XylE Part of K316007
- designed by Ben
Forward:
5’ TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCaacaaaggtgtaatgcgac 3’
Reverse:
5’ TATTCTGCAGCGGCCGCTACTAGTATTAACCGGTggtcagcacggtcatg 3’
J33210: LacZ
- designed by Brian
Annealing Temperatures: Forward=57.74°C Reverse=55.05°C
Forward:
5' TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCaccatgattacggattcac 3'
Reverse:
5' ATAACTGCAGCGGCCGCTACTAGTATTAACCGGTtcactccagccagc 3'
GFP and Flag Tag from K316007
- designed by Alex
Annealing Temperatures: Forward=56.88°C Reverse=59.95°C
Forward:
5' TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCcgtaaaggagaagaacttttc 3'
Reverse:
5' AATACTGCAGCGGCCGCTACTAGTATTAACCGGTcttgtcgtcatcatctttataat 3'
Sensors
Purify PCR Products
- J23113
- J23105
- J23118
- J23100
Miniprep
- B0051
Restriction Digest (vectors with EcoRI and XbaI)
- B0034+C0051
- B0015
Restriction Digest (inserts with EcoRI and SpeI)
- J23113
- J23105
- J23118
- J23100
- B0034+Mcherry
Ligations
- J23113 and B0034+C0051
- J23105 and B0034+C0051
- J23118 and B0034+C0051
- J23100 and B0034+C0051
- B0034+Mcherry and B0015
Transformations/Plating
All ligations
PCR
- B0030
- B0031
- B0032
- B0033
Wednesday
Reporters
The reporters then designed primers for site-directed gene mutagenesis for the XylE gene in order to eliminate the restriction sites from the XylE gene. These are the primers:
Note: Red font shows the mutagenesis site
Mutation 1: bp315 NgoMIV Site I
Annealing Temperature: 79.49°C
Original Sequence:
5' GGC CGG CGC GTG CGC TTC C 3'
Forward:
5' GGC CGA CGC GTG CGC TTC C 3'
Reverse:
5' G GAA GCG CAC GCG ATCG GCC 3'
Mutation 2: bp 486 XylE NgoMIV Site II
Annealing Temperature: 70.54°C
Original Sequence:
5' C GAC GAA TTG CCG GCG ACC TAT GAC C 3'
Forward:
5' C GAC GAA TTG CCC GCG ACC TAT GAC C 3'
Reverse:
5' G GTC ATA GGT CGC GGG CAA TTC GTC G 3'
Mutation 3: bp 837 XylE Agel
Annealing Temperature: 79.16°C
Original Sequence:
5' CAC AAA CCG GTG ACC TGG ACC ACC G 3'
Forward:
5' CAC AAA CCC GTG ACC TGG ACC ACC G 3'
Reverse:
5' C GGT GGT CCA GGT CAC GGG TTT GTG 3'
- The reporter group also began assembly of a construct featuring the XylE gene (with RBS) and a lac inducible promoter. This construct will be used to test the reporting system before the sensor system is created. Ben and Alex transformed the Plac and the XylE gene. The Plac part is R0010 and the XylE part is called J33204.
Sensors
Colony PCR
- B0034+Mcherry+B0015 (Kan resistance)
- B0034+Mcherry+B0015 (Amp resistance)
Purify PCR Products
- B0030
- B0031
- B0032
- B0033
Restriction Digest (vector with EcoRI and XbaI)
- C0051
Restriction Digest (inserts with EcoRI and SpeI)
- B0030
- B0031
- B0032
- B0033
Agarose Gel Electrophoresis
Chose colony A from B0034+Mcherry+B0015 from Kan plate
Ligations
- B0030+C0051
- B0031+C0051
- B0032+C0051
- B0033+C0051
Transformations/Plating
All above ligations Plated on AMP
Culture
- B0034+Mcherry+B0015 (kan) colony A
- J23113+B0034+C0051 (Amp) colony A
- J23105+B0034+C0051 (Amp) colony A