On Apr 3, 2012 3:45 PM, "elegans" <tashant@gmail.com> wrote:
>
> Hi again Nathan,
>
> Briefly: K. lactis is a yeast, not bacteria. Homologous recombination is easy in yeast, you can transform directly with PCR product and do not require viruses. As far as I recall, you can only hope to obtain pt mutations in
I haven't heard of that personally, can't see how it would only result
in point mutations either... seems like all or nothin
bacteria using this procedure directly... feel free to try it in
yogurt yeast though. Think about it. If you could just PCR amplify
anything and put it into bacteria without having to worry about
cloning plasmids, then why isn't everyone doing it?
Because you can't do a miniprep on genomic DNA during cloning operations.
Using the old googlulator, I found a paper transforming lactobacillus
acidophilus NCFM via homologous recombination using no viruses which
showed integration with as little as 169bp of homology on either end
of the insert, and stability over 35 generations. They used 500ng
plasmid DNA (from their cloning operations) 12.5kV/cm electroporation
and some special buffers... which could probably be done without/DIYed
http://www.scribd.com/doc/87924827/Appl-Environ-Microbiol-2011-Douglas-7365-71
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