Re: [DIYbio] Re: Bacillus Subtilis integration vector

On Mon, Jul 16, 2012 at 6:49 AM, Mega <masterstorm123@gmail.com> wrote:
> This here sounds great,
>
> http://ijb.nigeb.ac.ir/index.php/ijb/article/view/276/165
>
>
> Do E.Coli Promotors, ribosome bindingsites etc. match to B. subtilis?

Sometimes they work, sometimes they don't. To test this, you use a
promoter-cloning plasmid, which has origins or replication for
both/multiple species and a MCS (multiple cloning site) in front of
the gene (antibiotic resistance usually) and see which swapped-in DNA
allows expression (and thus resistance.. bacteria stays alive). The
swapped-in DNA is the 'promoter'/DNA to be tested.

The general rule is that a gram positive promoter is more likely to
work in another gram positive organism, and a gram negative promoter
is more likely to work in another gram negative organism:
http://www.sciencedirect.com/science/article/pii/0378111986903550

Here are some references from an old lab I did, where I fused pPL608
with pUC9 and found that the cat86 gene was expressed in both E.coli
and B.subtilis
https://docs.google.com/document/d/1kV1kfLPY__ZJdGUgmNqG3fSkawgo7zBUUe6OzNwbKo4/edit

2. WILLIAMS D.M., DUVALL E.J., LOVETT P.S. 1981. "Cloning Restriction
Fragments That Promote Expression of a Gene in Bacillus subtilis". J.
Bacteriology, June;146(3):1162-65

3. Keggins K.M., Lovett P.S., Duvall E.J. 1978. "Molecular cloning of
genetically active fragments of Bacillus DNA in Bacillus subtilis and
properties of the vector plasmid pUB110". PNAS, March;75(3):1423-27

4. Duvall E.J., Williams D.M., Mongkolsuk S., Lovett P.S. 1984.
"Regulatory regions that control expression of two
chloramphenicol-inducible cat genes cloned in Bacillus subtilis". J.
Bacteriology, June; 158(3):784–90

>
>
>
>
> Am Freitag, 13. Juli 2012 12:43:24 UTC+2 schrieb Mega:
>>
>> Hallo,
>>
>> Has anyone heard of a plasmid for integration in the chromosome of
>> b.subtilis?
>> If possible - that would be terrific - one that cuts out the antibiotic
>> resistance afterwards.
>>
>> I thought it would be a great idea to insert the lux genes into b.s.
>> because
>>
>> a) it's perhaps one of the most harmless bacteria.
>> b) it makes spores, so I can keep the petri dish when the sugars are gone,
>> dry it and some months later streak it on a new agar plate - still glowing.
>>
>>
>> Where do you get such a vector from? Do you have to build it yourself or
>> are there companies that sell it (just like pUC19, pVIB, pGreenII,...) ??
>>
>> Thx
>>
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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