Re: [DIYbio] Promising comp cell protocol for Bacillus

-----BEGIN PGP PUBLIC KEY BLOCK-----
Version: GnuPG v1.4.12 (GNU/Linux)
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=wOG4
-----END PGP PUBLIC KEY BLOCK-----
-----BEGIN PGP SIGNATURE-----
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Thunderbird - http://www.enigmail.net/
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=/D8L
-----END PGP SIGNATURE-----
> Anyway, on the mutant Bacillus, just grow it overnight at 30c in a
> liquid culture, then transfer it to 45c waterbath for like an hour.
> After that, treat it as an E coli culture and miniprep. It has a
> mutation in the PBSX prophage that causes it to lyse, and the phage
> doesn't package any of its own DNA. This is all in theory though,
> i am still creating the strains required.

As in, this is original work on your part? That looks really clever! One
of the hangups of working with bacillus is having to use Lysozyme for
minipreps. You can get it cheaply from brew-shops, but it's a pain all
the same. A method like this would be really handy if it worked reliably.

And yea, B.subtilis' competence system is pretty awesome. When you first
encounter it you think it's fairly straightforward, a DNA pump or
something. Then you look into it and find that it's this big, complex
and carefully orchestrated system for DNA uptake and active homologous
recombination.

There is precedent for using Bacillus as a "gibson assembly" platform;
IIRC, B.subtilis was used as the assembler for the first artificial
mitochondrial genome?

On 28/04/14 16:00, Koeng wrote:
> Not yet, but the "backbone" plasmid, pMK4, already works in Bacillus
> and I am just fusing the part into a lacZ fragment from the E coli
> shuttle vector side.
>
> Also I don't really have anything to purify plasmids from Gram
> positives yet... so I am just gonna make a new strain from a mutant
> Bacillus that lyses at 45c so you have to grow it at 30c. That way I
> can use kits that are used for E coli with the Bacillus. It would be
> cool if I can start putting Biobrick parts in the new plasmid then
> sporulating them, making it much easier to distribute the "registry".
> Instead of having to transform plasmids, just grow a stock from the
> sporulated cells. Or, better, the makers wouldn't have to maxiprep a
> ton of plasmids, just grow like 20mls for everyone. Anyway, on the
> mutant Bacillus, just grow it overnight at 30c in a liquid culture,
> then transfer it to 45c waterbath for like an hour. After that, treat
> it as an E coli culture and miniprep. It has a mutation in the PBSX
> prophage that causes it to lyse, and the phage doesn't package any of
> its own DNA. This is all in theory though, i am still creating the
> strains required.
>
>
> @Mega Lately I have read a bunch on natural competence, and let me
> tell you, Bacillus are freakin amazing at it. Not only do they import
> the dsDNA to a ssDNA, but they protect it from nucleases, then
> actually attach RecA to the strands and force it to go look in the
> chromosome for homology. I think that since they have gotten yeast
> transformations up to 10^6 I can do better with Bacillus. Integration
> is pretty common because of it, since the ssDNA is protected from
> everything and the Bacillus even keep a small "repository" of ssDNA
> strands to use in the short future when there is not enough RecA.
> Scientists have even transformed >100kb fragments at once even though
> Bacillus fragments the DNA into on average 18kb fragments
> (measurement was taken a long time ago, this size could possibly be
> because of DNA shearing. However, this doesn't explain the high
> transformation efficiency with small circular plasmids.) So Bacillus
> can integrate multiple fragments at once.... so I think I'll try a
> "gibson assembly" reaction once I get the plasmid working.
>
>
> On Monday, April 28, 2014 1:12:56 AM UTC-7, Cathal Garvey wrote:
>>
>> Promising stuff Koeng! Have you re-isolated the plasmid from the
>> cells, kochs-postulates-style, to show that they are transformed
>> successfully?
>>
>> On 27/04/14 19:50, Koeng wrote:
>>> Hey guys!!!
>>>
>>> I was tryna find an easier way to make Bacillus comp cells
>>> because I am very lazy. Here is what I did
>>>
>>> 1. Took about 10µl from the -80 freezer (yes I store Bacillus
>>> cells in a -80 freezer, don't judge) 2. Inoculated 3mls. I put in
>>> some xylose to express comK the master regulator of cell
>>> competence 3. Grew overnight. 4. Took 100µl and added 1µl of
>>> minipreped, circular DNA (I'll get concentration for ya'll when I
>>> go back to lab) The DNA has resistance to spec and was an
>>> integration vector into amyE. It also had a toxin, mazF, under an
>>> inducable promoter 5. Added some growth media (I was lazy and I
>>> think I added 100µl, not
>> sure
>>> just eyed-it) 6. Grew for 45 minutes in the 37c 7. Plated 50µl
>>>
>>> There is thousands of colonies (photo attached). I don't know if
>>> the
>> cells
>>> degrade the spec I selected with or something... but this is a
>>> lot of colonies. Anyone know details about spectinomycin? I
>>> thought I was gonna get about 10 colonies, but this is pretty
>>> outrageous.
>>>
>>> The one disadvantage of this: The cells grow visibly slower then
>>> normal. Overnight culture didn't give me that great of
>>> saturation.
>>>
>>>
>>> In the next week I think I'll improve this technique. It looks
>>> pretty promising though. I am also working on a Bacillus plasmid,
>>> kind of like what Cathal was doing, but with much less risk
>>> because I am just gonna
>> use
>>> pMK4 and fuse a Biobrick cassette in the PstI and EcoRi sites.
>>> Since
>> pMK4
>>> already works, hopefully it will work. BTW if anyone wants the
>>> plasmids
>> i
>>> work with or the strains i am using I'd be happy to send you
>>> them.
>>>
>>> -Koeng (Sorry the picture was too big to attach, here is a link
>>> to the photo) http://imgur.com/IEOL3T1
>>>
>>
>> -- T: @onetruecathal, @IndieBBDNA P: +353876363185 W:
>> http://indiebiotech.com
>>
>

--
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com