Berk2006-ConjugationTeam2

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MF&SIL_07/25/06

We have conjugated the pOX38 with some of Chris's stocks of pKD46 (in GeneHogs and MC1061) . We will have to see tomorrow if his stocks were still healthy.
Also, the trbCf complement PCR worked today (affirmed by a gel) and we have cleaned up the PCR, digested it in dPN1, EcoRI, and SpeI, then cleaned up the digestion, ligated with pSB1A2-I13511 digest in ecoRI and SpeI, and then transformed it into TG1 cells.
The sequencing data came back for the TraM and TraG complements and they are positive for both, so they are tough tagged and put in the freezer.
Finally, we are doing genomic minipreps of the RlambdaTraGknockout clones, from here on known as J23017. We then PCRed them with JL10 and JL11 oligos and hopefully when we map them, they'll show a band around 1200, instead of 2000 like last time, which indicated wild type Rlambda.
Here is the Rlambda traG KO PCR's Clones 6,7,8:

We are also adding double terminators from pSB1AKG0-B0015 digested in Eco/Xba to the TraM and TraG biobricks by digesting them in Eco and SpeI.

MF_07/24/06

Today we started cultures of pOX38 and pKD46 (in GeneHogs and MC1061). The pKD46 was from Chris's -80 stock. We are redoing the mating and the knockout because pOX38 mated with pKD46 aquired Cloramphenicol sensitivity somewhere in our procedure. Also, our four plates that we plated on Friday gave us unexpected results. All four plates came out with yellow colonies and smell like their is some kind of bacteria. We believe that DH10B is the culprit in this instance. We think that DH10B might have been contaminated before we began our experiment.
However, we are proceeding with the Rlambda TraG knockout by doing a genomic miniprep of the 4 cultures we grew up Friday, then PCRing with the TraG complement oligos JL10 and JL11, and finally mapping them to see if the size of the PCR fragment can give us a conclusion on whether TraG has been knocked out.
Also, the parts registry has been updated with all the complements and knockouts.
We are still waiting for out the sequencing data for the TraM and TraG complements.
We got our trbCf 20mers! So we are PCRing them with Olambda as the DNA template.
Mapping of cultures that we started on Friday was not successful. Gel electrophoresis showed three failures and one WT traGr. So, we are growing eight more over night cultures of J23017 [traGr] from Rlambda.