Reporter: Week 2 May 23-27

From 2011.igem.org

Revision as of 14:42, 8 June 2011

Monday

The reporters continued research on the linkers to hold the fusion proteins.

Tuesday

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.

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

     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'

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_lac and the XylE gene. The P_lac part is R0010 and the XylE part is J33204.

Friday

     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.