User:University of Washington/29 July 2008

luxR from pLac
-Bacterial stab of part I0462 received from iGEM HQ.

-Sequence of R0010+E0240 ligation received back and found to be inconclusive. Sequence will be submitted again, and assembly of these parts must be retried.

-Restriction digests of R0010 and E0240 started; will incubate at 37 overnight.

LuxR from AraC and TetR
-PCR purified the AraC and TetR promoter.

-Digest the Elowitz's plasmid with enzyme HindIII to find out what plasmid exactly the MG1655Z1 strain contain. Two potential >> pCS26 has 4 sites: expected 4716, 1990, 1982, 785 bp and pACYC184 has 1 site: 4245 bp
 * 34.5ul ddH2O + 5ul NEB2 + 10ul DNA(assume 100 ng/ul) + ...vortex... + 0.5 ul HindIII +....centrifuge ...
 * incubated 37 degree Celsius for 1.5 hours

-Ran gel. Saw two bands, one between 4 and 5 kb, the other between 500 - 1000 bp. The control(no enzyme) didn't have small fractions.

Lambda Red Recombineering of RP4 (Bryan)
PCR of recombinant vector containing CmC was successful! Purified PCR product.

PCR of recombinant vector containing TetC, probably because the colony lysate used as a PCR template had been sitting in the fridge for >1 wk. (Previous experiment had shown that colony lysate remained useful for several days.)

Rendered DY331 electrocompetent and aliquoted into glycerol stocks. Stored at -80 C. Will proceed with transformation and induction of recombination tomorrow.

AHL expression in yeast (Bryan)
Sequence of 2007 C0161 LuxI gene is confirmed.

Back to Team:University_of_Washington/Notebook