Re: [DIYbio] Re: Bacteria Lava-Lamp

Essentially, the problem is the flanking regions themselves. When
assembling a complex piece of DNA, you want to keep regular "save
points" in the form of transformed cells carrying the intermediate
steps. This is easy in E.coli and in B.subtilis with some basic design
considerations for most projects.

However, two of those design considerations for most species are:
1) Plasmid does not contain regions with strong homology to chromosome.
2) Plasmid does not contain highly repetitive regions.

If you build your integration vector in B.subtilis, you're breaking rule
#1. However, if you build it in E.coli, you're not, as there probably
won't be significant homology between the E.coli chromosome and the
B.subtilis elements you use to target integration.

That's why it's not an issue when you're just building an expression
plasmid without any aim of chromosomal integration; without the
repetitive regions, you don't need to worry about
accidental/premature/incomplete integration.

Of course, if you only assemble your plasmid in-vitro prior to use, you
don't need to use E.coli.. but your project may be more prone to failure
due to the number of backup-free steps involved in assembly. You could
try using a technique like gibson-assembly to mass compile all the
sub-units of your project together all at once, but I can't offer any
advice on implementation there, having never done it. :)

On 22/07/12 22:54, Chowe wrote:
> 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:
>>
>> 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.pdf
>>>
>>> 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.pdf
>>>
>>> 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
>>
>

--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey

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