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Reporter: Week 2 May 23-27
From 2011.igem.org
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| - | ==Monday== | + | ==Monday, May 23== |
The reporters continued research on the linkers to hold the fusion proteins. | The reporters continued research on the linkers to hold the fusion proteins. | ||
| - | ==Tuesday== | + | ==Tuesday, May 24== |
[[File:Jim_and_Alex_research.jpg||thumb|right|400px|Jim explains yet another mind-blowing paper on protein fusions to Alex.]] | [[File:Jim_and_Alex_research.jpg||thumb|right|400px|Jim explains yet another mind-blowing paper on protein fusions to Alex.]] | ||
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. | 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. | ||
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style="color:#FF0000">cttgtcgtcatcatctttataat</span> 3' | style="color:#FF0000">cttgtcgtcatcatctttataat</span> 3' | ||
| - | ==Wednesday== | + | ==Wednesday, May 25== |
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: | 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: | ||
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====Mutation 1: bp315 NgoMIV Site I==== | ====Mutation 1: bp315 NgoMIV Site I==== | ||
| - | '''Annealing Temperature:''' | + | '''Annealing Temperature:''' 84.32°C |
'''Original Sequence:''' | '''Original Sequence:''' | ||
| - | 5' GGC CGG CGC GTG CGC TTC C 3' | + | 5' GGC CGG CGC GTG CGC TTC C 3' |
'''Forward:''' | '''Forward:''' | ||
| - | + | 5' GTTGT GGC CG<span | |
| - | style="color:#FF0000"> | + | style="color:#FF0000">C</span> CGC GTG CGC TTC 3' |
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| - | + | ||
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====Mutation 2: bp 486 XylE NgoMIV Site II==== | ====Mutation 2: bp 486 XylE NgoMIV Site II==== | ||
| - | '''Annealing Temperature:''' | + | '''Annealing Temperature:''' 79.54°C |
'''Original Sequence:''' | '''Original Sequence:''' | ||
| - | 5' C GAC GAA TTG CCG GCG ACC TAT GAC C 3' | + | 5' C GAC GAA TTG CCG GCG ACC TAT GAC C 3' |
'''Forward:''' | '''Forward:''' | ||
| - | 5' C GAC GAA TTG CC<span | + | 5' C GAC GAA TTG CC<span |
| - | style="color:#FF0000"> | + | style="color:#FF0000">A</span> GCG ACC TAT GAC C 3' |
| - | + | ||
| - | + | ||
| - | + | ||
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====Mutation 3: bp 837 XylE Agel==== | ====Mutation 3: bp 837 XylE Agel==== | ||
| - | '''Annealing Temperature:''' 79. | + | '''Annealing Temperature:''' 79.38°C |
'''Original Sequence:''' | '''Original Sequence:''' | ||
| - | 5' CAC AAA CCG GTG ACC TGG ACC ACC G 3' | + | 5' CAC AAA CCG GTG ACC TGG ACC ACC G 3' |
'''Forward:''' | '''Forward:''' | ||
| - | 5' CAC AAA CC<span | + | 5' CCGGAC CAC AAA CC<span |
| - | style="color:#FF0000"> | + | style="color:#FF0000">A</span> GTG ACC TGG ACC ACC G 3' |
| - | + | ==Thursday, May 26== | |
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| - | ==Thursday== | + | |
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. | 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. | ||
| - | ==Friday== | + | ==Friday, May 27== |
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. | 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. | ||
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| + | [[Team:Penn_State/Notebook| Back to Notebook]] | ||
Latest revision as of 17:21, 13 July 2011
Contents |
Monday, May 23
The reporters continued research on the linkers to hold the fusion proteins.
Tuesday, May 24
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'
Wednesday, May 25
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: 84.32°C
Original Sequence: 5' GGC CGG CGC GTG CGC TTC C 3'
Forward: 5' GTTGT GGC CGC CGC GTG CGC TTC 3'
Mutation 2: bp 486 XylE NgoMIV Site II
Annealing Temperature: 79.54°C
Original Sequence: 5' C GAC GAA TTG CCG GCG ACC TAT GAC C 3'
Forward: 5' C GAC GAA TTG CCA GCG ACC TAT GAC C 3'
Mutation 3: bp 837 XylE Agel
Annealing Temperature: 79.38°C
Original Sequence: 5' CAC AAA CCG GTG ACC TGG ACC ACC G 3'
Forward: 5' CCGGAC CAC AAA CCA GTG ACC TGG ACC ACC G 3'
Thursday, May 26
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.
Friday, May 27
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.
