8/3/15 - Being as we moved from B. subtilis to E. coli we need to choose a new promoter+RBS for expression in E. coli. Part BBa_K608002 was then resuspended and transformed into NEB 5 alpha.
8/4/15 - Transformation was successful and colonies were picked to generate overnight cultures containing the constitutive promoter.
8/5/15 - All of the scar cloning materials finally came in the mail so we could begin trying to clone again. The overnight cultures were mini prepped and quantified using a NanoDrop. The GFP plasmid was digested with EcoRI and SpeI and the constitutive promoter was digested with EcoRI and XbaI. The digests were run on a gel, showing the appropriate sized bands. The desired bands were extracted with a gel extraction kit. The pieces were then ligated together at a 1:2 molar ratio and transformed into NEB 5 alpha cells.
8/6/15 - No colonies were observed on any of the plates indicating an unsuccessful transformation. The conclusion was that the DNA was exposed to the UV for too long and so we decided to run another gel using the restriction digests from before. Gel extraction was run quickly and then transformed.
8/7/15 - Still no colonies were observed from the previous transformation. Confident that it wasn’t a procedural error we turned the focus toward vector insert ratios. We reran the digests based on the same protocol as before but this time used a spectrum of molar ratios. We tried 1:1, 1:2, 1:5, and 1:10 vector to insert then ligated and transformed.
8/8/15 - No colonies were observed showing that the transformation was unsuccessful yet again. The hypothesis then turned to ligation time.
8/10/15 - Restriction digests were preformed based on the prior specifications. Gel followed by extraction showed proper bands. The ligation was then preformed for 2 hours at 16C to see if that would allow for proper ligation. The same spectrum of vector to insert ratios was used in the ligation. Samples were then transformed and plated.
8/11/15 - No colonies were observed again. With the cloning process taking as long as it is, we moved to trying to extract our regulator gene CFA. We requested a streak plate of E. coli K-12 from the Steven’s lab on the SCU campus to extract the DNA from.
8/13/15 - Plate was received from the Steven’s lab. Colonies were picked to generate overnights.
8/14/15 - A genome extraction was preformed on the overnights. DNA was then quantified using a NanoDrop. Primers were designed to flank the CFA synthase gene with overhangs that matched the sequence of the prefix and suffix.
8/17/15 - Received primers in the mail. PCRs were run on the genome using these primers. The product was then digested based on the standard assembly, as GFP was previously. CFA and GFP insert digests were gel extracted and ligated downstream of the constitutive promoter using the same spectrum of vector to insert ratios used previously. These products were then transformed and plated.
8/18/15 - Still no recombinants could be seen from the transformation. Thinking that we have changed all of the parameters that we could, we suspected that maybe there was an issue with the constitutive promoter sequence that was leading to neither of them working. Another promoter, BBa_J04500, was resuspended and transformed along with BBa_R0040, BBa_B0015, and BBa_B0034.
8/19/15 - Colonies were observed and picked to make overnight cultures.
8/20/15 - DNA was extracted from the overnights and glycerol stocks were made. The restriction digests were run as before. The digests were visualized on a gel and showed the appropriated sized bands. Gel extraction followed by ligation as before was conducted. The resulting DNA was transformed.
8/21/15 - No colonies were observed on the plates. Even though the bands were the right size, we decided to change the restriction digest run time to see if that would help. The digests were conducted for two hours as opposed to one. Visualized on a gel, extracted, ligated and transformed.
8/22/15 - No colonies were observed yet again. The only other step that we though could possibly be causing issues was the gel extraction step. This drops the efficiency of a ligation/transformation by several orders of magnitude. To fix this issue we changed around our gel extraction protocol so that each lane was precut out of the gel so that only the band being cut would be exposed to the UV opposed to all of them being exposed. Using this method we reran the standard assembly.
8/23/15 - Still no colonies were observed. With no other parameters to change we decided to switch back to scarless cloning, specifically PIPE. Primers pairs were designed to amplify both the CFA and GFP inserts as well as to open up the pLac promoter. Primers were then ordered.
8/25/15 - Primers arrived in mail. Primers were resuspended and used in PCRs with the GFP and pLac BioBricks along with the genomic DNA for CFA. Bands were generated for both CFA and GFP but not for pLac. The PCR for pLac was conducted again, this time with a gradient of annealing temperatures. The gel then revealed that we had a slight band in the right position. We then took that sample, along with CFA and GFP, and cleaned them in a PCR cleanup, quantified them and then ligated at a 1:2 molar ratio.
8/26/15 - Once again no colonies were seen on any of the plates. We then thought that maybe the ratio was not right. So we reran the DpnI digestion/ligation step of PIPE with the cleaned PCR products from before but with the previously used spectrum of vector to insert ratios. They were then transformed.
8/27/15 - No colonies were seen on plate.
8/31/15 - We reran the PCR on the pLac vector and this time saw much larger bands. The same PIPE procedure was conducted using this new DNA.