Re: [DIYbio] Bacillus: An alternative for E coli in DIYbio?

Bacillus subtilis can discard pieces of your DNA if the transformed
size if too large. An old source says:

"During transformation of B.subtilis, plasmid DNA is linearized as it
is transported into the cell. One strand of the entering duplex is
degraded, and resynthesized at a later step within the cell. Once
inside the cell, chromosomal DNA would recombine with homologous
sequences on the recipients chromosome. Plasmid DNA, however, has no
such homologous sites. Unless it is able to recircularize, plasmid DNA
is eventually degraded. No natural mechanism for rejoining linearized
plasmids has been described. Thus the only possibility for
recircularization is a self-recombination event between homologous
parts of a single plasmid or a multimeric plasmid."

"E. coli does not have a requirement for multimeric plasmids but will
generate multimers through the normal course of replication and
recombination."

"Therefore it is possible to initially transform E. coli with a
ligation mix and, on the following day, extract plasmid from the
transformed culture and use it to transform B. subtilis. A liability
with this strategy is the B.subtilis tends to randomly reduce the size
of large plasmids. Thus plasmids approaching about 10kb are likely to
suffer rearrangements and reductions."

pulled from:
Cloning Methodology Chapter 1a
Biotechnology Vol 7b 1989
http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/Cloning%20Methodology%20.pdf


On Wed, Jan 1, 2014 at 3:16 PM, Koeng <koen...@gmail.com> wrote:
> Hey guys!
>
> Recently I have begun using Bacillus subtilis a lot, and I gotta say... it
> is a LOT easier then E coli. For example I did one experiment with a strain
> of Bacillus that over expressed comK under a xylose inducible promoter...
> all I did was get the cells in growth phase, give them some DNA, grow them
> for 45 minutes then plate. I got transformation efficiency of 6x 10^3 with a
> circular integration plasmid. That is even easier then Cathal's Bacillus
> transformation protocol (no offense (:  ), which is one of the simplest out
> there. I can easily get better expression of comK by optomizing the RBS and
> I can make this work under  a sucrose inducible promoter! (Working on that
> right now) In addition to this, I have found a way to negatively select in
> Bacillus (I think it is the mazF gene from e coli), meaning that I could
> possibly make a strain which all you need to do is transform them, no
> selection method needed!
>
> Plus Bacillus can take pure PCR fragments... meaning that using homologous
> recombination and negative selection you could literally PCR an interesting
> protein from lets say, some cheek cells, then integrate it into bacillus.
> The integration could then be expressed, secreted, and then purified (using
> a variety of tags already in the bacillus, given that the gene has no
> introns/exons) Skip plasmids and e coli entirely! (of course... I need to
> make these strains)
>
> Anyone have anything to add or conflicting views?
>
> -Koeng
>
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--
-Nathan