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Team:UC Davis/Notebook/Week 12
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We embarked on the long process of characterizing our mutants this week. We're starting with the only ones that we have so far, the LacI promoters. The first priority with our characterization is to establish a good range of arabinose and IPTG concentrations form which we can get good data points. While cycling through these runs, we have been sure to screen other mutants (mainly C0012 for now).
--Monday 8/29/11--
--Wednesday 8/31/11--
Today marked the beginning of our characterization of R0010 mutants. We plan on characterizing six mutants at a time by running many 96-well plates (8 rows, 12 columns) through the Tecan. We will have six rows for mutants, one row for wild type, and a final row for our controls. Each plate will have a constant arabinose level in all wells (except the controls) and will have 4 levels of IPTG induction across each row. Not only will we be able to get a lot of data about how each mutant responds to various repressor concentrations, but we will determine a good range of arabinose and IPTG levels to test over in the upcoming weeks.
The first characterizing run will be for our "good mutants" #1-6 at 0% arabinose. The four levels of IPTG that we have decided to start with are 0 mM, 0.25 mM, 0.5 mM, and 1 mM.
--Thursday 9/01/11--
We are starting to gather data at a very fast pace. The current schedule is to let each plate incubate for either 6 hours at 37 °C or 18 hours overnight at room temperature, and then to run each plate for 6 hours in the Tecan at 37 °C. This allows us to get in three runs a day: one at 8:00 am, one at 2:00 pm, and one at 8:00 pm. Thankfully we have a hardworking team that is dedicated to keeping the Tecan running 24/7!
So far, we have been able to add runs with 0.1% arabinose, 0.01% arabinose, and another 0% arabinose (all at 0 mM, 0.25 mM, 0.5 mM, and 1 mM IPTG levels) to our list of completed runs.
--Friday 9/02/11--
Today we reevaluated the arabinose levels that we plan to use in our run. The original plan was to try different values on a logarithmic scale (0%, 0.001%, 0.01%, etc.), but the strain we are currently using (bw22826) shows a more linear reaction to various arabinose levels. We are instead going to try value between 0% and 1%, where we believe 0% to be minimal repression and 1% to be near maximal repression.
We have put in a 0.5% arabinose run (still with varying IPTG levels of 0 mM, 0.25 mM, 0.5 mM, and 1 mM) and are setting up a 0.75% and a screen for C0012 repressor mutants.
--Saturday 9/03/11--
It may be the weekend but the Tecan is still running! We've set up a 0% arabinose run and a 1% arabinose run for our R0010 mutants, both at 0 mM, 0.25 mM, 0.5 mM, and 1 mM.
--Sunday 9/04/11--
Today we are setting up and running more plates for the Tecan! We're still focusing on our R0010 mutants and are trying to establish a good range of arabinose concentrations for characterization. First, we are going to run another 0.75% arabinose plate, because our last one was did not turn out very well. We also plan on trying out a 2% arabinose run. We think that 1% arabinose is enough to fully induce the pBad promoter and give us near max repressor concentration, but doing a 2% run will confirm this for us. In addition to our characterizing runs, we'll also do another C0012 mutant repressor screen. At the moment, we're finding it somewhat difficult to screen for repressor mutants. We can't seem to produce much variation in the Tecan reads.
