Week 10

--Monday 8/15/11--

We got our sequences from last week in and checked them to make sure they looked good. Not all of the parts were correct, but we are about a day or two from being done with construction! Here is a table of which parts are completed and which parts we need to complete in order to finish construction.

In order to finish our construction, we are going to cut out R0010 and R0040 as inserts and put them in front of the parts needed. Tomorrow We will transform and hopefully have the finished construct by Wednesday.

Very few colonies on mutant ligation/transformation. We suspected that the BW22826 strain was not sufficiently competent, so we re-transformed ligation product into DH5a.

GFP mutants are clearly different. Made rep plates to better visualize differences.

Ligated and transformed yesterday's digestion.

Once again, their are quite a few colonies on our vector control plates. Fortunately, our ligation involved putting promoters (R0010 and R0040) in front of parts containing GFP (E0240+repressor). This means that a successful ligation/transformation will produce a green colony. There were no green colonies on our vector controls which indicated that any green colonies were successful ligations! We did a quick PCR screen and confirmed that this was the case. We now have the nine parts we need to screen and characterize our mutants!

Made rep plates of promoter mutants. Inoculated a 384-well plate with around 300 mutants to screen for promoter activity with GFP.

Yesterday we cultured our four completed construction so that we could miniprep them today. The plan now is to cut our three repressor screening constructions (Promoter+E0240+I13453+B0034) at Spe1 and Pst1. This will allow us to ligate these parts with our repressor mutants (cut at Xba1 and Pst1). The digestion of our construct looked very odd on a gel. We decided to set up the digestion again.

Got data back from the Tecan. Looks like we have a range of mutants!

We wrote a quick script to select 40 mutants with linearly-spaced fluorescence values. Using these mutants, we created rep plates and inoculated new 384-well plates for a second round of promoter screening, allowing five replicates for each promoter mutant. This should allow us to better assess the precision of the Tecan and the relative activity of the promoters.

Once again, the digest (Promoter+E0240+I13453+B0034) looked very odd when run on a gel. We observed either a weird gradient of bands instead of solid bands, or solid bands that are higher than the expected number of bases. We are suspicious of our enzymes, and have decided to set up a digest that will test their efficiency. We digested a random part 4 times with only one restriction enzyme in each digestion. Running this along side the uncut plasmid should tell us how well the enzyme is cutting. We got mixed results, but overall can conclude that our enzymes are working.

We set up the digestion again and will extract it from a gel on Monday.

We ran the inoculated 384-well quintuplicate screening plates to gather data on Saturday.

Quintuplicate mutant promoter screening of R0010 using the 384-well plate in the Tecan failed. Standard deviations for all mutants were extremely high. Under further inspection, it appeared that the wells of the plate received substantially different amounts of growth medium. Because of this, the optical density and fluorescence readings were both skewed, creating a large amount of variation in the data for our mutants, the wild-type control, our reference promoter (J23101) and our DH5-a control.

Because of this, we decided to abandon the use of 384-well plates due to their high contamination risk, the low sample volume and taxing time commitment to set up. Instead, we developed a Tecan schedule to implement over the next week that would allow multiple 96-well plates to be run in the same day. This should improve mutant screening throughput significantly.