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RecA: Week 3, May 30-June 3
From 2011.igem.org
Contents |
Monday, May 30
RecAI Extraction, Day 1
The RecA group extracted the RecAI gene from Escherichia coli using the genomic DNA extraction kit produced by Omega Bio-Tek. The RecA group created a culture of the RecAI gene and left this culture to grow overnight.
Tuesday, May 31
RecAI Extraction, Day 2
The RecA group performed miniprep on the RecAI culture from 5/30/11. The RecA group placed the RecAI gene into the C3 vector through amplified insert assembly in order to perform mutagenesis on the RecAI gene. This means restriction digest, followed by ligation, transformation into E. coli cells and plating to grow colonies over night.
RecA Mutagenesis Test, Day 1
The RecAI team also developed a plan for RecA mutagenesis. Figure 1 shows the overview of this plan. The following table describes each part in more detail.
| Part | Registry Name | Resistance | Position on Registry Plate | Length (bp) | Digested as (insert/vector) |
|---|---|---|---|---|---|
| 1: Terminator | B0015 | A/K | Plate 1 Well 23L | 129 | Vector |
| 2: Reporter (cI/RFP) | I763007 | A/A | Plate 1 Well 15J | 918 | Insert |
| 3: Constitutive Promoter | J23118 | A/A | Plate 1 Well 22A | 35 | Vector |
| 4: cI Repressor | K081007 | A/A | Plate 2 Well 20J | 796 | Insert |
The RecA group plans to insert RecA mutants downstream of the cI Repressor so that the constitutive promoter will continuously transcribe the cI Repressor and the RecA mutant. If the mutant has recombinant activity then the reporter will be deleted because of the homologous sequences of the Terminator (Part 1) and the terminator of the Reporter due to homologous sequences of the Terminator (Part 1) and the terminator of the Reporter, part 2 will be deleted.
Wednesday, June 1
RecAI Extraction, Day 3
The RecA group ran colony PCR for the RecAI+C3 constructs created 5/31/11. The agarose gel electrophoresis test showed that the assembly worked.
RecAI Mutagenesis Test, Day 2
The RecA group received their ordered parts from the registry and performed amplified insert assembly to add B0015 and I763007 together, as well as J23118 and K081007 together. This ended with allowing colonies to grow overnight on plates of the correct resistance.
Thursday, June 2
RecAI Extraction Take 2, Day 1
The RecA group realized that the RecAI+C3 colony dried up, so the group needed to make another one, starting with culturing RecAI from genomic DNA.
RecAI Mutagenesis Test, Day 3
The RecA group made cultures of the B0015+I763007 construct and the J23118+K081007 construct.
Friday, June 3
RecAI Extraction Take 2, Day 2
The RecA group miniprepped RecAI and assembled RecAI and C3 through amplified insert assembly.
RecAI Mutagenesis Test, Day 3
The RecA group prepared their assembly for sequencing by performing the miniprep procedure on the B0015+I763007 and the J23118+K081007 constructs. The RecA group then performed amplified insert assembly to combine the previous two constructs into one plasmid. The J23118+K081007 served as the insert and the B0015+I763007 served as the vector.
