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Team:UC Davis/Notebook/Week 14
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LacI promoter mutant characterization is moving forward at a steady pace; we have fully characterized mutants #1-5. It also seems that we have nine R0051 mutants and nine R0040 mutants as screening products that we can begin characterizing soon. The deadline for the first jamboree is approaching quickly, so we have also started working towards isolating our mutant LacI promoters for submission to the registry.
--Monday 9/12/11--
Today, we all regrouped to work out the plan for the week. Our primary concern now is to get R0040 and R0051 mutants before the deadline. Most of the screening results don't look very good, but we think that we can see a few mutants from each set. We're going to do another screen to see if we can possibly get good data from another screen of both R0040 and R0051 mutants. Tonight, when that run is over, we'll do a 1 mutant, 7 arabinose level, 4 IPTG level run for LacI mutant #4 (K611024) just to keep the Tecan busy. Tomorrow we'll look at this screening data and hopefully see some good results!
--Tuesday 9/13/11--
Today we're still passing the time by doing the 1 mutant, 7 arabinose level, 4 IPTG level runs for LacI mutant #7 (K611027). Aside from the Tecan, we had a very productive day. We've made just about everything we could with LB and chloro: LB+chloro plates, LB+chloro media, and LB+chloro+arabinose stocks at many different levels of arabinose.
--Wednesday 9/14/11--
Last night we did more of the extra-characterizing runs for LacI mutants #5 (K61105) and today just put one for #6 (K611026) in the Tecan. Unfortunately, it seems that something has gone wrong with our data the past few runs. The 0% arabinose level for all of our runs looks good, but everything from 0.2%-2% arabinose seems to fully repress our mutant promoters. Considering that the new LB+arabinose stocks that we made yesterday was carefully measured, there's a good probability that the old stock we were using (which was made before the summer) was mislabeled. Darn, just when things started getting on the right track again! It seems as though we're going to take a minor halt in our characterizing runs in order to calibrate our old data.
--Thursday 9/15/11--
In order to calibrate our old arabinose measurements, we're going to do serial dilutions of our new LB+chloro+arabinose stocks with known arabinose concentrations. We're starting with 0.2% arabinose and halving the concentration in each consecutive well. Each well will be inoculated with our the LacI wildtype construct, since we probably have the best understanding/characterization of this part so far. While analyzing this data we'll also do more runs with our R0040 and R0051 mutants.
Aside from Tecan runs, today was also a busy wetlab day. We began using PCR to get our 7 LacI mutants out of the characterization construct. Once we've done this, we'll ligate them back into pSB1C3 on their own so that they can be sent into the registry. We also took the time today to move our nine R0040 mutants and our nine R0051 mutants from the screening phase of our project to the characterization phase. We cultured them up and will miniprep and transform them into bw22826 tonight.
--Friday 9/16/11--
This morning we started another run that is significantly different from the runs we've been doing the past few weeks, an emission scan of our GFP mutants. We haven't done anything with these mutants since we generated them a while ago in order to test our mutagenic PCR protocol. We found some very interesting results though, such as an array of intensities of GFP and even a new orange variant of GFP that we have named OMGFP (orange mutated green fluorescent protein). Because we have some time between runs at the moment, we're going to try to see how these mutants behave.
