User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/04

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Prep for Heterologous Expression

 * Glycopeptide gene cluster has been sequenced which indicates that an additional clone may need to be recovered from the Utah library. There appear to be different tailoring and NRPS modules however, which shows that the pathway diverges from typical Class I-V glycopeptide backbone structures.  Due to a lack of selectable markers for the Rec/ET strategy used in the lab for more than three constructs.  I am gearing up to do a TAR cloning strategy where the inserts will first be temporarily shunted into a carrier vector with homing endonuclease sites on either side flanking the insert.  The freed insert will then be recombinationally cloned with other pieces using the yeast TAR strategy outlined by Steven Lory.


 * The primers for this strategy are being designed and will first target the inserts from our existing pWEB library in order to facilitate the recombination. These pieces will be designed with a secondary strategy in mind as well.  The in vitro recombination efforts pioneered by the JCVI in their Mycobacterium genome assembly paper will be tried as an alternative strategy in parallel.


 * BB16 clones from S.Lividans, Viridochromogenes, Albus (selected on MS + NA + Apramycin for one week at 30C). Were prepped for spore stock generation into 10% glycerol.  50ul of this stock was innoculated into 100ml of freshly made SMM in aerosol flasks.  Shaken at 200 RPM 30C for expression checking.  Ethyl Acetate extraction and HILIC separation will continue as described.


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