Bacillus Subtilis integration vector

[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

[Mega]

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?

[Nathan McCorkle]

On Mon, Jul 16, 2012 at 6:49 AM, Mega <masters...@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

[Nathan McCorkle]

On Fri, Jul 13, 2012 at 6:43 AM, Mega <masters...@gmail.com> wrote:
> Hallo,
>
> Has anyone heard of a plasmid for integration in the chromosome of
> b.subtilis?

a quick google of "subtilis homologous vector integration" comes up
with loads of answers
www.bgsc.org/Catpart4.pdf
www.bgsc.org/NewProducts/Middleton.pdf

and something a little closer to home:
http://partsregistry.org/Bacillus_subtilis
"Integration plasmids is the most used technique by specialist in B.
subtilis. They are E. Coli plasmids with two homologous sequence with
two fragments of a chromosomal gene of B. Subtilis, and a resistance
cassette inserted in between. If your brick is cloned in between these
two homologous region, the vector act as a shuttle that integrate your
construct into the chromosome."

[Andreas Sturm]

Yeah, but those don't have self-excising marker genes.

And I reguard it as highly unethical to play around with chromosomal integration of antibiotic resistances, because if one bug escapes (and in diy bio this may hypothetically happen), resistant (really resistant, not just plasmids they lose again without selective pressure!) germs will spread .... What if an anthrax bacterium (relative to b.subtillis, so gene fits) takes up the DNA and gets resistant?


Thanks for the link regarding promotor in gram + and -   ;)

 

2012/7/16 Nathan McCorkle <nmz...@gmail.com>

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[Nathan McCorkle]

On Mon, Jul 16, 2012 at 5:01 PM, Andreas Sturm <masters...@gmail.com> wrote:
> Yeah, but those don't have self-excising marker genes.

I don't think there is any system that is self-excising in use today
in the synbio world, it could probably be made using a stripped down
transposon type system though.

That said, if you integrate a marker, it doesn't have to be antibiotic
resistance. Even if it was, B. subtilis 168 is a lab strain, so it's
not likely to survive outside the lab. And it's just as easy to remove
the resistance yourself once you get your gene of interest in the
genome, you just knock it out using the same integration vector
technique.

[Cathal Garvey]

He's right to have _some_ concern about resistance, given that wild
relatives of B.subtilis are commonplace (same as E.coli, or most other
free-living mesophiles really) and pretty compatible with subtilis genes.

Of course this certainly fits into the usual "inappropriate use of
antibiotics by farmers/doctors does far more harm" argument, which is
absolutely true. But, all the same I think he's right to take
responsibility for his work.

As to self-excision.. if you can afford the DNA, it's easy. You just
place two LoxP sites in the correct orientation around the DNA you want
to delete, and include the Cre enzyme in that cassette with an inducible
promoter. When induced, the Cre enzyme will trigger excision of the
LoxP-flanked DNA. It will also trigger re-integration of this DNA, but
over time the DNA is more likely to be lost than retained as long as you
maintain the induction system for some generations.

So, your DNA would look like this:
--- Desired Gene ---LoxP--AmpR--Cre--LoxP---

Induction gives this:
--- Desired Gene ---LoxP---
And a circular cutout:
(=AmpR==Cre=LoxP=)

Eventually leading to just:
--- Desired Gene ---LoxP---

Orientation of LoxP sites is important: look up the system and learn how
it works. If you put the sites in the wrong orientation, you'll end up
with a rapidly flipping cassette, rather than one that cuts itself out
of the genome. That's cool and all, but totally useless!

LoxP is, if I recall, somewhat long: about 30bp. In a bacterial genome,
this means it's unlikely to occur by chance, so odds of massive genetic
upheaval due to Cre use are low. You could use any number of
bacteriophage integrases to do the same thing, using the non-identical
integration sites. Efficiency will be higher as the reaction is one-way,
but using Cre does have the advantage of leaving a LoxP site behind that
could be used later for integrating more interesting DNA.. :)

On 16/07/12 22:25, Nathan McCorkle wrote:
> On Mon, Jul 16, 2012 at 5:01 PM, Andreas Sturm <masters...@gmail.com> wrote:
>> Yeah, but those don't have self-excising marker genes.
>
> I don't think there is any system that is self-excising in use today
> in the synbio world, it could probably be made using a stripped down
> transposon type system though.
>
> That said, if you integrate a marker, it doesn't have to be antibiotic
> resistance. Even if it was, B. subtilis 168 is a lab strain, so it's
> not likely to survive outside the lab. And it's just as easy to remove
> the resistance yourself once you get your gene of interest in the
> genome, you just knock it out using the same integration vector
> technique.
>


--

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PGP Public Key: http://bit.ly/CathalGKey

[Andreas Sturm]

Synthesizing:
30 base pairs * 2 * 0,4$/bp = 24$.

They will clone it into any commercial vector, so I choose a b.subtilis plasmid. Maybe they will also clone also the amp resistance into it for ''a few bucks''.

So at all that wouldn't be below 100 $s? (minimum costs of synthesis are 100$ anyway)

2012/7/18 Cathal Garvey <cathal...@gmail.com>

[Mega]

Ah ok, with this Cre-Lox System you would rely on that the amp reistance gets sorted out. Or would you reccomend inserting a counter marker (makes it non-auxotrophic, but where to get minimal-medium as a home genetic engineer)?

But it is kind of inevitable that the resistance is erased after some time, I think?


Additionally. I was wrong. You shouldn't take a B.Subtilis plasmid because it will replicate. Best would be something like pUC18, you amplify it in E.Coli and then miniprep -> B.Subtilis. If one subtilis can propagate, it has a copy of the gene in its chromosome.