Weekly Journals

We have been keeping a daily journal of our progress in the lab after our initial brainstorming, lab training, and basic project preperation. Here is a compiled, weekly journal entry of our project.

Week 1 (May 31 - June 6)

Attempted to PCR amplify fragment 4 and vector pCR 2.1. Initial attempt with COWg4 failed, nothing showed up at the 1 kb line when ran through the gel. PCR products of Vector 2.1 showed up at exactly the 1 kb line when run through the gels. We suspected the annealing temperature was too low and was the cause of these issues. The new PCR products of COWg4 were run through the gel, but all of the products were smaller than 50 bp.

Week 2 (June 7 - June 13)

Performed four more PCRs on COWg4 and designed primers for gibsoning COWg fragments into pET21 expression vector, as well as gibsoning COWg fragments into their individual cloning (PCR 2.1) vectors. First chemical transformation of the COWg4-pCR2.1 plasmid into the TOP10 cells failed due to contaminated SOC media. Second chemical transformation completed but not optimal. Newly designed primers arrived and made into 100 uM and 20 uM working stock. Also made 1:10 dilutions of COWg1, COWg2, and COWg3. Ran four PCR reactions for each fragment. Then created more 1% TAE gels to run 10 uL of each PCR product against a 100 bp ladder. COWg1 and COWg2 worked well, COWg4 was ambiguous, and COWg3 failed and showed up around 500 bp (should be roughly 1200 bp). Performed PCR clean-up on remaining products that were not run through the gel and nanodropped. Did minipreps on ON cultures. Gibsoned all four fragments into cloning vectors, then chemically transformed into TOP10 cells.

Week 3 (June 14 - June 20)

Sequence-verified COWg4 in PCR 2.1, with clones 4 and 5 having 100% perfect sequences. Transformation of COWg1 was successful and made into 5 ON cultures. ON cultures were miniprepped and sent into for sequencing. Sequencing data revealed that the primers did not bracket the COWg1 sequence correctly, resulting in two short fragments of COWg1 with nothing in between. Transformation of COWg2, COWg3, and COWg4 were unsuccessful. Redid transformation of COWg2, plated and incubated overnight for colony growth. Designed new primers for COWg3. Tried to use gibson assembly again for COWg1 using the new pCR 2.1 vector from the PCR earlier in the week, and then transformed into TOP10 cells. Miniprepped COWg2 using ON cultures.

Week 4 (June 21 - June 27)

Performed more transformations of COWg1 and started four ON cultures with it. COWg1 cultures were then miniprepped and sent in for sequencing, which failed and resulted in many unidentifiable tags (extra basepair somehow designed into primer, and thus added at the start and end of each sequence after PCR amplification). Made a new COWg1 primer that should avoid previous issues. Made ON cultures of COWg2 due to the low yields of the minipreps of COWg2, possibly due to leaving it resuspended in P1 buffer over the weekend. Minipreps of COWg2 from eight colonies were finished, with two being sent for sequence verification, both of which failed. Sent in new COWg2 fragments with M13 primers this time for sequencing. Ran COWg3 through a gel, purified, and transformed it. Redesigned primers to amplify the vector for COWg3.

Week 5 (June 29 - July 4)

Sequencing results for the COWg2 fragments with the M13 primers failed, resulting in only 41 bp of COWg2. Started a new Gibson assembly on COWg3 and transformed the finished product, and amplified Vector 2.1. Ran EcoRI experiment with COWg1 and COWg2. Started COWg1 from the beginning. We sequenced the COWg1 and COWg2 PCR products without the vector and it looks like we have a shortened product we’re putting in there.

Week 6 (July 5 - July 11)

Received sequencing results with a lot of “N”s. We then mixed methanol and aspirin to create the Wintergreen scent, which proved to be very strong. Completed the transformation for COWg1 and created ON cultures. COWg1 was then miniprepped and sent in for sequencing with the M13 sequencing primers, which were later found to have the wrong sequencing primers. We redid the PCR amplification and gel purification of COWg2 and transformed COWg3. Both COWg2 and COWg3 were then put separately into the Gibson assembly with Vector 2.1 and then transformed into the TOP10 cells. Later in the the week we took more ON cultures of COWg2 and COWg3 and miniprepped it to send it in for sequencing, using M13 primers.

Week 7 (July 12 - July 18)

Received sequencing results for COWg2 and COWg3. COWg2 had many errors while the COWg3 fragments fell just short of the acceptable range of errors. We then picked 10 colonies from COWg3 and made ON cultures, which subsequently miniprepped and sent in for sequencing. Two of the clones were sequence verified. Also designed and ordered Quikchange primers for COWg1. We met with Dr. Davies to figure out how to make an anaerobic chamber for carbon monoxide testing, and chose a beta radiation storage box. We received a culture of pCDF-gfp from Drew, which we miniprepped to get the pCDF-gfp plasmid. Got back the primers for replacing T7 with pCoof in pCDF for PCR? PCR products of pCDF were run through a gel and extracted through purification. A second PCR was done for pCDF because the first one had inadequate purity. The Gibsoned COWg1 PCR 2.1 was transformed into TOP10 cells. A new PCR was done for COWg2. New ON cultures were made for COWg1, and were miniprepped and sent in for sequencing. pCDF-pCoof-gfp was transformed into TOP10 and DE3 cells, with ON and liquid cultures respectively.

Week 8 (July 19 - July 25)

Sequencing results for PCR 2.1- COWg1 came in; three were perfect except for one terminator. Did pCoof- gfp transformation into TOP10 cells with ON cultures. Primers for COWg1 were redesigned and ordered without the extra T7 promoter and any extra terminators. SPEC plates were made and the anaerobic chamber was constructed in the Physics machine shop.

Week 9 (July 26 - August 1)

Overnight cultures of DE3 cells and cog1 PCR 2.1 were made. The team electrotransformed COWg1 pcr2.1 into DE3 cells. Next, the team designed and ordered pET21 primers and plated COW1 pcr 2.1 DE3 on the appropriate AMP and KAN plates. The following day, pCoof-gfp version 4 for sequencing prior to 10 am when sequencing products are picked up. Sequencing results for pCoof-gfp version 3 arrived and didn’t look optimal. Sequencing results also arrived for COW1_FES and tubes 2 and 3 were sequence verified. PCR reactions were set up for all four fragments, although when the products were run through the gel, only COWg3 worked which was then gel extracted. The team also checked the plates of COWg1 pcr 2.1 DE3 (on KAN plates) although there were no colonies formed thus they were left in the incubator and a new plate of COWg1 pcr 2.1 DE3 were plated onto KAN plates and allowed to grow in the incubator. The team also transformed and plated COWg1_pcr 2.1 DE3 + pCoof-gfp v.4 #1 (on SPEC/KAN plates). pCoof-gfp version 5 underwent PCR clean-up. The following day, part of the team participated in the bake sale to raise money for the competition. pCoof-GFP version 4 had 1 correct clone, 1 with 3bp deletion, and 9 T7-GFP clones. COWg1 PCR 2.1 DE3 + pCoof-GFP version 4 #1 had many colonies. The team also transformed pCoof-GFP #8 into COWg1 PCR 2.1 DE3 electrocomp cells at a 1:10 and 1:100 dilutions. The team also transformed pCoof-GFP #8 and COWg1 FES into DE3 chemcomp cells. The team abandoned pCoof-GFP version 5. Since only the PCR for COWg3 worked previously, the team redid the PCR for cog1, 2, and 4. COWg3 was gel purified. The following day, the team checked the dilution, although no colonies had grown. The team also realized that COWg1_FES DE3 was plated on wrong antibiotic plate (SPEC instead of AMP) and that pCoof-gfp DE3 was plated on wrong antibiotic plate (AMP instead of SPEC). With this mistake in mind, the team plated COWg1 FES DE3 (chem comp), 100 uL, onto KAN plates + 2x YT and pCoof GFP version 4 #8 DE3 (chem comp), 100 uL, onto SPEC plates. DE3 COWg1 PCR2.1 + pCoof-GFP version 4 #8 was also plated onto SPEC/KAN plates. The following day, more SPEC, KAN, and SPEC/KAN plates were made as with 1% TAE gels. There were colonies for pCoof-gfp v. 4 #8 DE3, COW1_FES DE3, although no colonies for COW1_pcr2.1 DE3 + pCoof-gfp. Gels were run for the PCR products of COW1, 2, 4 and only 1 worked and 2 appeared but was at the wrong length. They were both extracted and purified. The team redid the PCR of COW2 and pet21 the following day. Restriction enzyme digest was also done.

Week 10 (August 2 - August 8)

This week consisted primarily of making COW1_FES DE3 ON cultures. The cells were made electrocomp and pCoof-gfp was transformed into them. The week also was replete with meeting with Dr.Ellington and outlining the trajectory of the project. The team also redid the PCRs for COW2 and pet21 DE3 + pCoof-gfp and after repeating several times.. The were also gel purified. The team also began testing with the DE3 cells, T7 cells, T7 pCoof cells, and the pcoof cells. The team used 100 uM of CORM when testing as prescribed in the papers referenced. The chamber was set up in the chemical hood and the team flushed the chamber with argon gas to remove and residual oxygen.

Week 11 (August 9 - August 15)

Testing proceded this week. The team continued to use 100 uM of CORM. Fluorescence readings were taken and recorded. The setup didn’t change at all, rather the team looked to replicate the data that they had previously acquired in the week prior.

Week 12 (August 16 - August 22)

The team continued to redo the PCRs for pet21 and COW2 and finally received fruitful results. The restriction digest was done. The team ligated pCoof_RBS into pSB1C3 plasmid backbone and transformed into TOP10 cells (chemically transformed) and plated on a Cam (Chloramphenicol) plate. Also, more 1% TAE gels were made. Three ON cultures of COW1 pCoof were made. The fluorescence of COW1 pCoof was measured and experimentation proceeded. More cultures were made for experimentation. A mini experiment took place to find the optimal amount of CORM to add to the suspended cultures during experimentation. The team found that the initial 100 uM concentration was optimal. two trials were able to completed this week with the 100 uM concentration of CORM.

Post-Experiment Phase (August 23 - September 18)

The data from fluorescence readings was analyzed and graphed. Poster, parts submissions, and wiki page completed.

Team:LASATX/Notebook