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From 2011.igem.org

Contents 1 Monday 1.1 Reporters 1.2 Sensors 1.3 All 2 Tuesday 2.1 Reporters 2.1.1 Primers 2.2 Sensors 2.2.1 Purify PCR Products 2.2.2 Miniprep 2.2.3 Restriction Digest 2.2.4 Ligations 2.2.5 Transformations/Plating 2.2.6 PCR 3 Wednesday 3.1 Reporters 3.1.1 Mutation 1: bp315 NgoMIV Site I 3.1.2 Mutation 2: bp 486 XylE NgoMIV Site II 3.1.3 Mutation 3: bp 837 XylE Agel 3.2 Sensors 3.2.1 Colony PCR 3.2.2 Purify PCR Products 3.2.3 Restriction Digest 3.2.4 Agarose Gel Electrophoresis 3.2.5 Ligations 3.2.6 Transformations/Plating 3.2.7 Culture 4 Thursday 4.1 Reporters 4.2 Sensors 4.2.1 Sequencing 4.2.2 PCR 4.2.3 Restriction Digest 4.2.4 Ligation 4.2.5 Transformation/Plating 5 Friday 5.1 Reporters 5.2 Sensors 5.2.1 Agarose Gel Electrophoresis 5.2.2 Corrections for next week 5.2.3 Confirmed and Named Ligations

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

1. J23113
2. J23105
3. J23118
4. J23100

Miniprep

     B0051

Restriction Digest

• Vectors with EcoRI and XbaI

1. B0034+C0051
2. B0015

• Inserts with EcoRI and SpeI

1. J23113
2. J23105
3. J23118
4. J23100
5. B0034+Mcherry

Ligations

1. J23113 and B0034+C0051
2. J23105 and B0034+C0051
3. J23118 and B0034+C0051
4. J23100 and B0034+C0051
5. B0034+Mcherry and B0015

Transformations/Plating

All ligations

PCR

1. B0030
2. B0031
3. B0032
4. 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 P_lac and the XylE gene. The P_lac part is R0010 and the XylE part is J33204.

Sensors

Colony PCR

1. B0034+Mcherry+B0015 (Kan resistance)
2. B0034+Mcherry+B0015 (Amp resistance)

Purify PCR Products

1. B0030
2. B0031
3. B0032
4. B0033

Restriction Digest

• Vector with EcoRI and Xba

     C0051

• Inserts with EcoRI and SpeI

1. B0030
2. B0031
3. B0032
4. B0033

Agarose Gel Electrophoresis

Chose colony A from B0034+Mcherry+B0015 from Kan plate

Ligations

1. B0030+C0051
2. B0031+C0051
3. B0032+C0051
4. B0033+C0051

Transformations/Plating

All above ligations were transformed and plated on Amp resistant plates.

Culture

1. B0034+Mcherry+B0015 (kan) colony A
2. J23113+B0034+C0051 (Amp) colony A
3. 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 P_lac 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

1. B0034+Mcherry+B0015
2. J23113+B0034+C0051
3. J23105+B0034+C0051

• all verified by sequencing

PCR

     B0034+Mcherry+B0015

Restriction Digest

• Vector with EcoRI and XbaI

1. J23113+B0034+C0051
2. J23105+B0034+C0051

• Insert with EcoRI and SpeI

     B0034+Mcherry+B0015

Ligation

1. B0034+Mcherry+B0015 and J23113+B0034+C0051
2. 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

1. B0034+mCherry+B0015 => K648000
2. J23113+B0034+C0051 => K648001
3. J23105+B0034+C0051 => K648002

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