User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/05

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Heterologous expression and other thoughts

  • SMM strep cultures of BB16 are rocking at 30C.
  • R5 media being prepped for het expression of same constructs
  • Yeast Capture primers ordered for amplifying pLLX8 (CYH2, bla) and pLLX13 (yeast capture, ura 3, tetR) for ZB118. We should be able to use the downstream homology region for designing the upstream capture vector for the overlapping clone ZA41. Targeted a 1kb and 2kb retion (tREG and ABC transporter) of ZB118 for homologous recombination.
  • Basic outline will follow where we subclone the inserts into the yeast capture vectors, check insert size with iSceI digestion, and proceed with the TAR cloning strategy outlined by S. Lory. Advantages: does not require selectable markers (a problem with pathways greater than 2 clones).
  • Waiting for primers for Q87 overlap screening.