Week 2: May 23-27
From 2011.igem.org
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<blockquote>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.'''</blockquote> | <blockquote>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.'''</blockquote> | ||
- | <blockquote>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 | + | <blockquote>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 undetermined stimulus.</blockquote> |
- | <blockquote>Dr. Richard also proposed a | + | <blockquote>Dr. Richard also proposed a modelling 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.</blockquote> |
===Tuesday=== | ===Tuesday=== | ||
====Reporters==== | ====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 | + | 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 designing primers for PCR. We needed primers for the XylE part, the linker, the tev cleavage site and the GFP/flag tag complex of the Imperial College part, K316007, the CI repressor/RecA cleavage site, the LacZ part (J33210), the K243004 linker, and the K105012 linker. |
=====Primers===== | =====Primers===== | ||
Note: | Note: | ||
- | <blockquote> <span style="color:#04B404">Green font indicates junk DNA ends (binding sites).</span> </blockquote> | + | <blockquote> <span style="color:#04B404">Green font indicates junk DNA ends (binding sites).</span></blockquote><blockquote><span style="color:#FF0000">Red font indicates annealing sites.</span></blockquote><blockquote>CAPITAL LETTERS INDICATE PREFIX AND SUFFIX.</blockquote><blockquote>lowercase letters indicate gene of interest. </blockquote> |
- | + | ||
- | <blockquote> <span style="color:#FF0000">Red font indicates annealing sites.</span> </blockquote> | + | |
- | + | ||
- | <blockquote> CAPITAL LETTERS INDICATE PREFIX AND SUFFIX. </blockquote> | + | |
- | + | ||
- | <blockquote> lowercase letters indicate gene of interest. </blockquote> | + | |
- | + | ||
'''Linker used in K316007''' | '''Linker used in K316007''' | ||
Line 181: | Line 174: | ||
#J23118 | #J23118 | ||
#J23100 | #J23100 | ||
- | |||
=====Miniprep===== | =====Miniprep===== | ||
- | + | B0051 | |
- | =====Restriction Digest | + | =====Restriction Digest===== |
+ | *Vectors with EcoRI and XbaI | ||
#B0034+C0051 | #B0034+C0051 | ||
#B0015 | #B0015 | ||
- | + | *Inserts with EcoRI and SpeI | |
- | + | ||
#J23113 | #J23113 | ||
#J23105 | #J23105 | ||
Line 195: | Line 187: | ||
#J23100 | #J23100 | ||
#B0034+Mcherry | #B0034+Mcherry | ||
- | |||
=====Ligations===== | =====Ligations===== | ||
#J23113 and B0034+C0051 | #J23113 and B0034+C0051 | ||
Line 202: | Line 193: | ||
#J23100 and B0034+C0051 | #J23100 and B0034+C0051 | ||
#B0034+Mcherry and B0015 | #B0034+Mcherry and B0015 | ||
- | |||
=====Transformations/Plating===== | =====Transformations/Plating===== | ||
All ligations | All ligations | ||
Line 214: | Line 204: | ||
====Reporters==== | ====Reporters==== | ||
- | The reporters | + | The reporters 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 | Note: Red font shows the mutagenesis site | ||
Line 271: | Line 261: | ||
- | 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 P<sub>lac</sub> and the XylE gene. The P<sub>lac</sub> part is R0010 and the XylE part is | + | 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 P<sub>lac</sub> and the XylE gene. The P<sub>lac</sub> part is R0010 and the XylE part is J33204. |
====Sensors==== | ====Sensors==== | ||
Line 283: | Line 273: | ||
#B0032 | #B0032 | ||
#B0033 | #B0033 | ||
- | =====Restriction Digest | + | =====Restriction Digest===== |
- | + | *Vector with EcoRI and Xba | |
- | + | C0051 | |
+ | *Inserts with EcoRI and SpeI | ||
#B0030 | #B0030 | ||
#B0031 | #B0031 | ||
Line 300: | Line 291: | ||
=====Transformations/Plating===== | =====Transformations/Plating===== | ||
All above ligations were transformed and plated on Amp resistant plates. | All above ligations were transformed and plated on Amp resistant plates. | ||
- | |||
=====Culture===== | =====Culture===== | ||
#B0034+Mcherry+B0015 (kan) colony A | #B0034+Mcherry+B0015 (kan) colony A | ||
Line 309: | Line 299: | ||
====Reporters==== | ====Reporters==== | ||
- | Alex and Jim carried out miniprep, ran PCR on the XylE gene (insert), and digested the XylE gene with XbaI and PstI and the P<sub>lac</sub> using SpeI and PstI. Ben and Brian carried out ligation, transformation and plating. The reporter group also ordered | + | Alex and Jim carried out miniprep, ran PCR on the XylE gene (insert), and digested the XylE gene with XbaI and PstI and the P<sub>lac</sub> using SpeI and PstI. Ben and Brian carried out ligation, transformation and plating. The reporter group also ordered the catechol substrate to use with the Imperial College reporting system (K316007). |
====Sensors==== | ====Sensors==== | ||
Line 318: | Line 308: | ||
*''all verified by sequencing'' | *''all verified by sequencing'' | ||
=====PCR===== | =====PCR===== | ||
- | + | B0034+Mcherry+B0015 | |
+ | |||
=====Restriction Digest===== | =====Restriction Digest===== | ||
*Vector with EcoRI and XbaI | *Vector with EcoRI and XbaI | ||
Line 324: | Line 315: | ||
#J23105+B0034+C0051 | #J23105+B0034+C0051 | ||
*Insert with EcoRI and SpeI | *Insert with EcoRI and SpeI | ||
- | + | B0034+Mcherry+B0015 | |
+ | |||
=====Ligation===== | =====Ligation===== | ||
#B0034+Mcherry+B0015 and J23113+B0034+C0051 | #B0034+Mcherry+B0015 and J23113+B0034+C0051 | ||
Line 331: | Line 323: | ||
*the above ligations were transformed and plated on Amp resistant plates | *the above ligations were transformed and plated on Amp resistant plates | ||
+ | ===Friday=== | ||
+ | ====Reporters==== | ||
+ | The reporter group ran colony PCR on the created colonies then ran the results on a gel. The gel showed that the construct consisted of less than 500 base pairs, but should have been over 1100 base pairs. We think that only the lac inducible promoter showed up in our colonies. As a result of this set back, we will have to redo assembly next week. | ||
+ | ====Sensors==== | ||
+ | =====Agarose Gel Electrophoresis===== | ||
+ | B0034+Mcherry+B0015 | ||
+ | *''The results of the gel showed that the assembly worked.'' | ||
+ | |||
+ | =====Corrections for next week===== | ||
+ | The sensor group must redo the two B0034+mCherry+B0015 and constitutive promoter+B0034+C0051 ligations as well as the B0030/B0032 + C0051 ligations. | ||
+ | =====Confirmed and Named Ligations===== | ||
+ | #B0034+mCherry+B0015 => K648000 | ||
+ | #J23113+B0034+C0051 => K648001 | ||
+ | #J23105+B0034+C0051 => K648002 | ||
Latest revision as of 15:50, 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 undetermined stimulus.
Dr. Richard also proposed a modelling 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 designing primers for PCR. We needed primers for the XylE part, the linker, the tev cleavage site and the GFP/flag tag complex of the Imperial College part, K316007, the CI repressor/RecA cleavage site, the LacZ part (J33210), the K243004 linker, and the K105012 linker.
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
- 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 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 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 Xba
C0051
- 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 were transformed and plated on Amp resistant plates.
Culture
- B0034+Mcherry+B0015 (kan) colony A
- J23113+B0034+C0051 (Amp) colony A
- J23105+B0034+C0051 (Amp) colony A
Thursday
Reporters
Alex and Jim carried out miniprep, ran PCR on the XylE gene (insert), and digested the XylE gene with XbaI and PstI and the Plac using SpeI and PstI. Ben and Brian carried out ligation, transformation and plating. The reporter group also ordered the catechol substrate to use with the Imperial College reporting system (K316007).
Sensors
Sequencing
- B0034+Mcherry+B0015
- J23113+B0034+C0051
- J23105+B0034+C0051
- all verified by sequencing
PCR
B0034+Mcherry+B0015
Restriction Digest
- Vector with EcoRI and XbaI
- J23113+B0034+C0051
- J23105+B0034+C0051
- Insert with EcoRI and SpeI
B0034+Mcherry+B0015
Ligation
- B0034+Mcherry+B0015 and J23113+B0034+C0051
- B0034+Mcherry+B0015 and J23105+B0034+C0051
Transformation/Plating
- the above ligations were transformed and plated on Amp resistant plates
Friday
Reporters
The reporter group ran colony PCR on the created colonies then ran the results on a gel. The gel showed that the construct consisted of less than 500 base pairs, but should have been over 1100 base pairs. We think that only the lac inducible promoter showed up in our colonies. As a result of this set back, we will have to redo assembly next week.
Sensors
Agarose Gel Electrophoresis
B0034+Mcherry+B0015
- The results of the gel showed that the assembly worked.
Corrections for next week
The sensor group must redo the two B0034+mCherry+B0015 and constitutive promoter+B0034+C0051 ligations as well as the B0030/B0032 + C0051 ligations.
Confirmed and Named Ligations
- B0034+mCherry+B0015 => K648000
- J23113+B0034+C0051 => K648001
- J23105+B0034+C0051 => K648002