This week’s update is a little bit different because the team has split into sub teams to be most effective in getting our project to completion. For now we have a group working on the killswitch part of our project and a team working with the G blocks of our enzymes. After the killswitch team completes their part, they will rejoin the rest of the group and work to take the project to completion
Monday, July 6th
Kill switch: completed a mini prep of the RBS, lysing agent, promoter and terminator with amazing results (above 300ng/ul) on the nanodrop. They also revamped the protocol for digestion and assembly to hopefully yield better results than the previous attempts
Enzyme: Spent the morning doing gel extractions and then testing their results on the nanodrop. Currently in a cycle of PCR, gel electrophoresis, gel extraction, then nanodrop. If the nanodrop results are less than 10 ng/ul repeat the cycle! All 14 G blocks need to make the cut so it is a lengthy process.
Tuesday, July 7th
Kill switch: a new ligation protocol was implemented today which had a lot more incubation time on the digestion (4 hrs) and ligation (16 hours). During our down time today we plated many things so that we had extra stocks of parts that we have already transformed this summer.
Enzyme: After repeating the cycle that we were in on Monday, we had some success! Our BLANK BLANK G Blocks had concentrations above our goal of 10ng/ul. And then we got back to the cycle so that all our G Blocks are above that 10ng/ul threshold.
Wednesday, July 8th
Kill Switch: Today we transformed the Promoter with RBS and the Lysing agent with the terminator into E. Coli making our first dual part transformation! Hopefully we’ve had enough practice and it was successful. We also made a plethora of AMP plates because we were low. Lots of waiting around while things incubate and autoclave today.
Enzyme: Today we continued to PCR, run gels, image, extract, and test the concentrations of DNA with the nanodrop. We were able to get started with Gibson Assembly on the G Blocks that had concentrations of 10ng/ul or above from Tuesday!
Thursday, July 9th
Kill Switch: The dual part transformation from Wednesday wasn’t successful. After a short mourning period we decided to re- ligate the digestion from Tuesday using some more tips from Sam and Janie. Today was also filled with more house-keeping items, like autoclaving lots of equipment, and making broth for inoculations.
Enzyme: Electrophoresis gels were run on the Gibson assembly that was tried yesterday. Unfortunately, this Gibson was not successful. A practice yeast transformation was also performed.
Friday, July 10th
Kill Switch: Turns out that our transformations were successful! They just needed extra time to grow. Unfortunately, we already had ligated and used competent cells, so we decided to plate them anyway to see if the tips from Sam and Janie yielded better results.
Enzyme: After yesterdays failed Gibson assembly, we tried to do it again. Second times the charm, this time we used Sam as a resource and used ethanol precipitate to get rid of the elution buffer so that we could save the DNA.