User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/16

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TAR cloning

  • Capture vector 1 was selected for PmeI digestion and linearization. Digest was performed as per 25 ul standard conditions in buffer 4+BSA as per NEB protocol with 0.5ul of enzyme for 2 hrs 37C.
  • ZB118 linearized with HindIII digest for 1hr using same conditions and both reactions were heatkilled at 70C for 15 minutes.
  • Yeast competent cells were made from 5ml YPD O/N growth at 30C. 1:100 dilution into 50ml x 2 YPD for 5 hours, then washed 40ml H2O (1200xg 5 min spins) and resuspended in freshly made TFBI (100mM LiAc, 1xTE, 500ul)

Heterologous Expression

  • Cultures still need some time to grow (S.Virido, S.Lividans). EtAc extractions again did not show appreciable differences in profiling over a 30min gradient of Acetonitrile 80-0 on C18. The phosphinothricin group may be a #*($ to protonate even in acidic conditions so a metabolomics HILIC hydrophilic column characterization and separation scheme is being hatched at the moment. (looking at Kyu Rhee's work)