PROJECT

RESULTS

Day 29 - Monday, July 25th 2011

Ran out the digested 3 part ligations on a gel to confirm what colonies contain what parts. The gel showed that our 3A ligations worked when they produced clear colonies. The red colonies, as we suspected, contained the original backbone+RFP. Our 2A ligations were a failure, with no bands in the expected range. We think the small pieces we cut out of the backbone during digestion may have religated.
Retransformed the 3A ligations (CP -> RBS -> Receptors) from which we did not get clear colonies the first time. We will also try religating in case the ligation was the problem.
Miniprepped the “clear” samples from last night’s overnights.
Made glycerol stocks of the “clear” parts.
Started overnight cultures of our “clear” CP -> RBS -> RFP colonies so we can plate them and analyze fluorescence.
Confirmed that our sequenced registry part matches the receptor genes from the pseudomonas aerugionsa genome.
Looked into travel grants.

Day 30 - Tuesday, July 26th 2011

Met with the high-throughput facility at Northwestern that may help us characterize our promoters.
Started digesting yesterday’s minipreps in order to confirm that all of our samples were successful.
We had no colonies on the retransformations from yesterday, so we religated the CP -> RBS -> LasR/RhlR parts
Planned the testing process for analyzing our promoters.

Day 31 - Wednesday, July 27th 2011

Miniprepped the overnights of the new colonies from our RBS34 ligations. Once again, most of the DNA concentrations were extremely low. We ran the 3 best samples on a gel to try and determine if these colonies were the correctly ligated part, or if our entire ligation had failed.
Started a digest of our 2 part Assemblies to run them on a gel and attempt to determine if they are correct.
Also started religating all of the RBS34 ligations.
Overnighted colonies from the glycerol stock plates of our four successful RBS30 3 part ligations.
We analyzed our CP-> RBS -> GFP on a UV plate. There was no flourescence. However, this was not surprising because these were the same parts that did not appear correct on the gel yesterday.
Several team members were trained on the plate reader at the High Throughput Analysis Laboratory.
Plated glycerol stocks of GFP, RFP and some other parts which we are running low on DNA.

Day 32 - Thursday, July 28th 2011

Miniprepped overnights from our successful glycerol stock parts and the clear colonies from our CP -> RBS -> Receptor retransforms
Ran out a gel of the retransforms to see if these clear colonies would give us the correct parts
PCRed some basic parts that we were running low on such as LasR and GFP.
Transformed two constitutive promoters so that we can start from scratch if necessary
Designed and ordered oligonucleotides containing the constitutive promoter that we can ligate directly.
Discussed strategies with our advisors for proceeding with the testing phase of our project.
Overnighted RBS and ligations.

Day 33 - Friday, July 29th 2011

Did PCR clean-up of yesterday’s PCRed parts
Miniprepped RBS overnight
Miniprepped, digested, and ran gels of ligation overnights for confirmation purposes
Made more SOB
Overnighted constitutive promoters - have decided to give a different one from the Registry a shot
Ordered constitutive promoter oligos