RecA: Week 4, June 6-11



Monday, June 6

RecAI Extraction Take 2, Day 3

All of the colonies that grew on the plates were purple, meaning that RecAI did not successfully insert into the C3 vector. Thus, a third attempt at RecAI extraction began with extracting the RecAI gene from the Escherichia coli genome and created a culture to let grow over night.

RecAI Mutagenesis Test, Day 4

According to the sequencing, instead of B0015+I763007 and J23118+K081007, the RecA group made B0015+J23118 and I763007+K081007.

Tuesday, June 7

RecAI Extraction Take 3, Day 1

The RecA group performed a third amplified insert assembly to insert RecAI into the C3 vector.

RecAI Mutagenesis Test Take 2, Day 1

The RecA group sent the parts from the registry (B0015, I763007, J23118, K081007)to sequencing to make sure that these parts are correct. RecA carried out amplified insert assembly to create the B0015+I763007 and J23118+K081007 constructs. A mistake labelling resulted in the following possible constructs:


Wednesday, June 8

RecAI Extraction Take 3, Day 2

The RecAI colonies were purple again, but the sensor group had similar problems. The RecAI digest from 6/7/11 was ligated with the K3 digest created by the sensor group. The ligation was transformed into Escherichia cells and plated onto Kan resistant plates.

RecAI Mutagenesis Test Take 2, Day 2

Colonies for B0015+I763007, B0015+K081007, J23118+I763007 grew, but no colonies for J23118+K081007 grew overnight. The proposed reason that J23118+K081007 did not grow is poor transformation, as shown by the poor time constant measured when transforming. This ligation was redone using the digests from 6/7/11, and was transformed and plated to let colonies grow overnight.

The B0015+I763007, B0015+K081007, J23118+I763007 colonies were amplified through colony PCR, but did not show up in the agarose gel electrophoresis. Even after both the colony PCR and gel electrophoresis were repeated, the colonies did not show up. The colonies were cultured overnight anyway. The possible reason the colony PCR did not work is the DNTP's were mislabelled, preventing amplification.

Thursday, June 9

RecAI Extraction Take 3, Day 3

The RecAI+K3 colonies were run through colony PCR and the products were put through agarose gel electrophoresis. No bands showed on the gel, but many groups in the lab experienced this problem. The cause is likely a mislabelling of the dNTPs in the lab. The colonies were placed in a culture and grown overnight.

RecAI Mutagenesis Test Take 2, Day 3

The B0015+I763007, B0015+K081007, J23118+I763007 constructs were miniprepped and sent to sequencing. The J23118+K081007 construct did not grow again. Sequencing results came back:

constitutive promoter+PcI reporter
labeled correct because promoter
exists in front of reporter.
terminator+cI repressor
only terminator (repressor got "lost")
terminator+PcI reporter
turned out to be terminator+repressor (wrong labeling)

Final Sequencing Conclusions: The RecA group believes that contamination occured in the repressor and reporter (K081007 and I763007) parts, so these parts were transformed from the registry into Escherichia coli cells and left to culture overnight. Since the terminator and constitutive promoter (B0015 and J23118) were verified through sequencing, freezer stock of these parts were prepared.

Friday, June 10

RecAI Mutagenesis Test Take 2, Day 4

     The PcI reporter (I763007) and the cI repressor (K081007) did not grow in the culture prepared on 6/9. Thus, the RecA group transformed these parts again into Escherichia coli cells from the registry. The reporter and cI repressor parts from the registry were also used to perform amplified insert assembly. The ampicillin resistant plates were left overnight to allow colonies to grow.

     The constitutive promoter (J23118) and the terminator (B0015) were miniprepped in preparation for assembly of the entire construct. This assembly will consist of placing the promoter + terminator piece with the cI repressor (K081007) + reporter (I763007) piece into one construct.

Saturday, June 11

RecAI Mutagenesis Test Take 2, Day 5

     No PcI reporter (I763007) + cI repressor (K081007) cells grew on the ampicillin plates from 6/10. The ligation from 6/10 (PcI reporter (I763007) + cI repressor (K081007)) were transformed into Escherichia coli cells in an attempt to grow cells on ampicillin plates again with the hope that this time would produce colonies.

     The RecA group discovered today that the sensor group had successfully made a construct containing a constitutive promoter in front of the cI repressor. This part has been sequenced and sent to the registry. This part can be used for the RecAI mutagenesis test construct.

     Amplified insert assembly was performed on the terminator (B0015) and the reporter (I763007), and the assembly was transformed into Escherichia coli cells and plated onto ampicillin resistant plates.

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