Team:UC Davis/Notebook/Week 4

BW22826 Cells had very fast growth overnight, and were too overgrown to use for competent cells in the morning. The procedure was restarted late at night (around 9:30) and lesser amounts of starter culture were used (1, 2, and 2 mL) to ensure the cells don't overgrow. One culture was placed on a shaker at room temperature, the other two were placed on a bench at room temperature.

We inspected our LacI mutants. Most of the plates were covered in white colonies, though a few had some slightly pink colonies, indicating mutations were successful. The cells were plated on IPTG+ plates, so we expect induction; it is possible that the mutation rate was high and the promoter function was destroyed in a majority of colonies. Ten pink and ten white colonies were replated on IPTG+ plates, to rule out poor induction on our initial plates.

E0240 variants with B0034 and with no RBS in pSB1K3 were cultured.

--Wednesday 7/06/11--

The making of our competent cells was successful! We were able to produce two full boxes today, with good amounts of cells in each of the tubes. Hopefully we won't have to make more for a while.

We also miniprepped the E0240 variants from yesterday so that we could proceed with construction. We digested these minipreps along with our three main promoters (R0040, R0051, R0010) so that we could ligate them together.

Today started slow but ended in a flurry! We ligated three promoters (R0010, R0040, R0051) to E0240 (GFP) and transformed them. We also retransformed our mutant LacI ligations to try and get even more. The previous batch of potential mutants which were different shades of red on a replica plate got replated to see if we'll be able to see a clearer difference between shades of red on streaked plates. So far there appears to be a good range of redness which is a positive sign that our mutagenic pcr worked somewhat well. Out of 12 plates, we got 9 possible mutants. We're planning on taking a closer look with our Tecan plate reader early next week. This will give some more definitive proof of good mutants or not. These mutants also got cultured so we can prepare them for sequencing soon.

In the Dh5α strain, LacI repressor is always present so, upon induction we expected to see red. It seems from our mutation protocol that we mutated them too heavily since most colonies were white even with IPTG present.

Mutated C0012, C0051, C0040. Ligations of promoters to GFP looked green=good! Made control constructs for R0010 Ligated R0040 and R0051 mutants into promoter screening plasmid for screening!