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RecA: Week 5, June 13-18
From 2011.igem.org
Contents |
Monday, June 13
RecAI Mutagenesis Test, Take 2 Day 6
No colonies grew over the weekend. Thus, the PcI reporter (I763007) was assembled to the terminator (B0015) through amplified insert assembly. The assembly hit a road block at the plating stage because no agar plates existed in the lab. Thus, the RecA group attempted to make plates themselves. The transformed cells were grown in culture, but the recovery media with the cells were kept in order to plate the next day, once more plates were made.
The testing construct will be able to use either part K648001 or K648002 parts, which were assembled and sequenced by the sensor group. These parts feature the constitutive promoter in front of the cI represser, which is necessary for the RecA1 mutagenesis testing construct.
Tuesday, June 14
RecAI Mutagenesis Test, Take 2 Day 7
The assembly from 6/13 was plated early in the morning in order for the colonies to grow all day and be ready for colony PCR at night. Unfortunately, the colonies did not grow again.
RecAI Extraction Take 4, Day 1
In order to store the RecAI mutant strain in a plasmid, the RecAI part was inserted into the K3 vector since the C3 vector did not work. Ampified insert assembly was used for this insertion. The RecAI PCR product was purified and digested with XbaI and PstI. The K3 miniprep from 6/8 was digested with XbaI and PstI. The two digests were ligated together, and the ligation was transformed into Esherichia coli cells and plated onto kanamycin resistant plates.
Wednesday, June 15
RecAI Mutagenesis Test, Take 2 Day 8
The plates featuring cells containing the assembly from 6/14 contained no colonies. The PcI reporter (I763007) and the terminator (B0015) pieces from the registry were transformed into Escherichia coli cells and were left in culture overnight.
RecAI Extraction Take 4, Day 2
The only colonies that grew overnight were red, indicating that the RFP gene was not replaced by the RecAI gene. Thus, the restriction digests made on 6/14 were ligated then transformed into Escherichia coli cells and plated onto kanamycin plates. The purified RecAI PCR products were sent to sequencing to ensure that the RecAI extraction worked.
Thursday, June 16
RecAI Mutagenesis Test Take 2, Day 9
DNA from the cultures created on 6/15 containing the terminator (B0015) and the PcI reporter (I763007) was extracted through the Omega Bio-Tek miniprep protocol. The PcI reporter insert was amplified through PCR, and the product was purified. Since the registry DNA containing the PcI reporter and the terminator parts was running low, freezer stock was made of the two parts for both the -80°C and the -20°C freezers.
One proposed hypothesis for the continuous failed assemblies was the restriction enzymes have died. For this assembly, XbaI and SpeI enzymes from Dr. Salis' lab were used for restriction digest. SpeI was used instead of PstI because Dr. Salis did not have PstI in his lab. Dr. Salis also suggested doing longer restriction digest (4-5 hours instead of 1-2 hours) in order to obtain more DNA.
Friday, June 17
RecAI Mutagenesis Test Take 2, Day 10
Amplified insert assembly was performed in another attempt to connect the terminator (B0015) to the PcI reporter (I763007). The restriction digests were left in the 37°C water bath for three hours instead of the usual two hours in order to allow more time for the reaction to take place. The digests were ligated, transformed into Escherichia coli cells and plated onto ampicillin resistant plates.
RecAI Extraction Take 5, Day 1
The purified RecAI PCR product from 6/7 was digested for two hours with XbaI and PstI. The K3 miniprep from 6/8 was digested with the same restriction enzymes for two hours. The RecAI and K3 digests were ligated together, transformed into Escherichia coli ciells and plated onto kanamycin resistant plates.
Saturday, June 18
RecAI Mutagenesis Test Take 2, Day 11
No colonies grew on the plates again, so plating the recovery media containing the cells was performed again on kanamycin plates.
RecAI Extraction Take 5, Day 2
The only colonies that grew for this assembly were red, indicating that the RecAI gene did not successfully insert into the K3 plasmid in the RFP gene location.
