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

Hmm, one of the references there is:
http://diyhpl.us/~nmz787/pdf/DNA_Shuttling_Between_Plasmid_Vectors_and_a_Genome_Vector_Systematic_Conversion_and_Preservation_of_DNA_Libraries_Using_the_Bacillus_subtilis_Genome_BGM_Vector.pdf

which does actually mention multiple month growth and heat cycling and
spore induction with tests before and after.

I found that they use BReT to get the DNA back out, but couldn't find
a nice overview of that which didn't require me to read too much more.

On Fri, Jan 3, 2014 at 10:33 AM, Koeng <koeng101@gmail.com> wrote:
> I think if you have the resources to transfect cells that it would be easy
> to get plasmids
>
> @nathan Thats for plasmids though. I am talking about homologous
> recombination into the genome. It is actually more stable then any other
> large DNA cloning method, given that you do it properly. It can stably hold
> 3Mbps, and most likely more. Also, using plasmids in Bacillus for homologous
> recombination would be rather unstable, since 1 untransformed plasmid could
> destroy your experiment
>
> http://www.pnas.org/content/102/44/15971.full.pdf
>
> http://www.researchgate.net/publication/6489230_Direct_cloning_of_full-length_mouse_mitochondrial_DNA_using_a_Bacillus_subtilis_genome_vector
>
> http://www.biomedcentral.com/1471-2164/14/300
>
>
> On Friday, January 3, 2014 10:18:51 AM UTC-8, Nathan McCorkle wrote:
>>
>> 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
>
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--
-Nathan

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