I have found a site that offers two different plasmids that express the lux operon (one for gram-positive one for gram-negative) http://www.caliperls.com/products/reagents/in-vivo-imaging-reagents/light-producing-cells-and-microorganisms/plasmids/.
Thanks for the info Cathal. Why would you build the plasmid for E.Coli? Since E.coli is gram-negative, couldn't I build my plasmid in B.subtilis then PCR amplify the construct out of the plasmid? Im just thinking that it would save me needing an additional plasmid, since I will already have the B.Subtilis vector to get the flanking regions. I would cut out the gram-positive modified lux operon from the plasmid that I found in the link above. Insert it into a B.Subtilis vector that contains large chromosomal flanking regions for B.subtilis. Then PCR amplify my construct and transform. I could try both ways, plasmid DNA and the linear DNA since I will already have a plasmid constructed.
On Sunday, July 22, 2012 5:08:21 PM UTC-4, Cathal wrote:
On Sunday, July 22, 2012 5:08:21 PM UTC-4, Cathal wrote:
Just an additional FYI:
Getting DNA into E.coli works best with supercoiled plasmid DNA, but
getting DNA into B.subtilis works best with linear DNA.
The reason is that B.subtilis uses an active uptake system to absorb DNA
in the environment, which can only absorb DNA if it can find a loose end
to chew on. It's been speculated that most B.subtilis transformants from
plasmid solutions are actually those that found damaged or cut plasmids
and absorbed them, which contributes to B.subtilis' undeserved
reputation for being genetically unstable (that is, the DNA was already
damaged, it's not the bug's fault! :))
The differences in efficiency are pretty dramatic. Especially because
the active uptake system includes chaperone proteins that help trigger
homologous recombination with the chromosome if there are suitably
homologous regions.
So, my suggestion is the build the DNA you want in a shuttle plasmid,
with nice big (500bp+) chromosomal flanking regions, in E.coli. Then use
PCR to generate loads of linear copies of the desired flanking regions
and genes, and transform with PCR product. You'll *probably* get better
results that way.
That's only true if you're trying to get chromosomal integration. For
plasmids in B.subtilis, efficiencies are pretty good with lab strains,
so you can ditch E.coli and just work with B.subtilis if you have the
right plasmids and workflow.. an area I'm working on right now myself.
Protip: if you're designing DNA for B.subtilis 168 derivatives, avoid
the XhoI restriction enzyme site, and the TopoI recognition site for
B.subtilis: WCATWTAWWA (W = A/T, acc. to IUPAC DNA notation). You'll get
considerably higher efficiency and DNA stability.
On 22/07/12 19:08, Andreas Sturm wrote:
>> Also how exactly would you build a plasmid that would insert part of its
> genes into the chromosome, any certain plasmids need to be used?
> http://www.bgsc.org/Catpart4.
>
> Here are some plasmids listed. It is written that on request, they may send
> you a plasmid for free if you use it for non-profit things.
>
> Well, the plasmid needs two flanking sites that fit to a gene in the Bac.
> Subt. Chromosome. Then in 1 out of 10000 cases, homologous recombination
> occurs. The plasmid does not replicate, so if the bacillus doesn't die from
> the antibiotic and forms a colony, it has the gene casette integrated.
>
> In theory, you could also use pUC19, fit in flanking sites of B.Subt.
> (where to get the template?, can you design the primers?), inside the
> flanking sites Lux operon + Resistance , amplify it in E.coli, transform
> B.S.
>
>> I will have full time access to my schools laboratory.
>
> Awsome ;)
>
>> For that project would you recommend B.Subtilis?
>
> Yeah, because of the spore formation. E.Coli will die on the plates after a
> while when the sugar is out. After at last one year, you cannot revive them
> again.
> The spores of B.S., when they 'smell' better conditions, will 'wake up'
> again and in theory, still after 50 years of dryness, etc. you'll get a
> glowing B.Subt. again.
>
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
On Sunday, July 22, 2012 5:08:21 PM UTC-4, Cathal wrote:
Just an additional FYI:--
Getting DNA into E.coli works best with supercoiled plasmid DNA, but
getting DNA into B.subtilis works best with linear DNA.
The reason is that B.subtilis uses an active uptake system to absorb DNA
in the environment, which can only absorb DNA if it can find a loose end
to chew on. It's been speculated that most B.subtilis transformants from
plasmid solutions are actually those that found damaged or cut plasmids
and absorbed them, which contributes to B.subtilis' undeserved
reputation for being genetically unstable (that is, the DNA was already
damaged, it's not the bug's fault! :))
The differences in efficiency are pretty dramatic. Especially because
the active uptake system includes chaperone proteins that help trigger
homologous recombination with the chromosome if there are suitably
homologous regions.
So, my suggestion is the build the DNA you want in a shuttle plasmid,
with nice big (500bp+) chromosomal flanking regions, in E.coli. Then use
PCR to generate loads of linear copies of the desired flanking regions
and genes, and transform with PCR product. You'll *probably* get better
results that way.
That's only true if you're trying to get chromosomal integration. For
plasmids in B.subtilis, efficiencies are pretty good with lab strains,
so you can ditch E.coli and just work with B.subtilis if you have the
right plasmids and workflow.. an area I'm working on right now myself.
Protip: if you're designing DNA for B.subtilis 168 derivatives, avoid
the XhoI restriction enzyme site, and the TopoI recognition site for
B.subtilis: WCATWTAWWA (W = A/T, acc. to IUPAC DNA notation). You'll get
considerably higher efficiency and DNA stability.
On 22/07/12 19:08, Andreas Sturm wrote:
>> Also how exactly would you build a plasmid that would insert part of its
> genes into the chromosome, any certain plasmids need to be used?
> http://www.bgsc.org/Catpart4.
>
> Here are some plasmids listed. It is written that on request, they may send
> you a plasmid for free if you use it for non-profit things.
>
> Well, the plasmid needs two flanking sites that fit to a gene in the Bac.
> Subt. Chromosome. Then in 1 out of 10000 cases, homologous recombination
> occurs. The plasmid does not replicate, so if the bacillus doesn't die from
> the antibiotic and forms a colony, it has the gene casette integrated.
>
> In theory, you could also use pUC19, fit in flanking sites of B.Subt.
> (where to get the template?, can you design the primers?), inside the
> flanking sites Lux operon + Resistance , amplify it in E.coli, transform
> B.S.
>
>> I will have full time access to my schools laboratory.
>
> Awsome ;)
>
>> For that project would you recommend B.Subtilis?
>
> Yeah, because of the spore formation. E.Coli will die on the plates after a
> while when the sugar is out. After at last one year, you cannot revive them
> again.
> The spores of B.S., when they 'smell' better conditions, will 'wake up'
> again and in theory, still after 50 years of dryness, etc. you'll get a
> glowing B.Subt. again.
>
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
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