Friday, July 24, 2009

Update from Circuit World

Hi, this is Emily again! I'm part of the wetlab portion of our team and today I'm going to be updating you on the progress we've made over the past week or so. I'll start with the signalling circuit where Jeremy, Jamie and Carol have been concentrating their efforts.

This week, Carol has been working on our promoter library which Jeremy touched on last week. Essentially Carol is trying to find a promoter that provides the best expression of Luciferase so that we can find the optimal expression of LuxPQ. We don't know the optimal expression of PQ however as more epression of LuxPQ does not necessarily mean more expression of Lucierase. This is therfore the purpose of the Promoter Loibrary, to find the optimal expression of LuxPQ. To do this, Carol has set up PCR reactions with 16 forward primers and 16 reverse primers. She then digested the PCR products and the Surette vector with ZhoI and BamHI enzymes and is currently in the process of ligating them in order to trasnform them into competant cells. She has tried three different types of Ligation, QuikLigse, T4 NEB ligase and a week long ligase. The first two did not produce cells that glow (that express Luciferase) and there were not many cells that grew. The week long ligation will be done tomorrow and we are hoping that the results will be promising!

Last week Jamie and Jeremy finished the signalling circuit with LuxOU and LuxPQ. This week they have been working on a plasmid switch. Thw signalling circuit was contructed in an AK vector, however we need to now move it into a vector with K resitance (the surrette vector). Because moving something from AK to K does not offer any antibiotic selection pressure, we need to first do a plasmid switch to get it into an AC vector, and then we can move it into the Surrette vector.

Next we have the reporter circuit which Kevin has been working on. This week Kevin has been making a constrcut with pqrr4, RBS and GFP in order to test the functioning of the Signalling circuit. Kevin has been working on this construction this week and is waiting for

Finally we have our two Mutant circuits: LuxOD47E and LuxOD47A, which are being worked on by Emily and Vicki respectively. As of last week, Vicki had seccessfully completed and sequnced her circuit. This week she has been working on her pro paper that, with some additons and changes is well on its way to being done. Unfortunately, LuxOD47E and I are not having so much fun and I am pretty much at the same place that I was last week, trying to get the B0015 terminator in. This week's attempt seems much more promising as yesterday I did a verificatioon digest with goiod results and have since sent off a colony for sequncing. I have my fingers crossed that B0015 is in there and my circuit will finally be done.

Well that's about all for this week. We still have a fair ways to go in the lab, so hopefully we have more successes to report next week!

No comments:

Post a Comment