Promising comp cell protocol for Bacillus

[Koeng]

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

[Cathal Garvey]

Promising stuff Koeng! Have you re-isolated the plasmid from the cells,
kochs-postulates-style, to show that they are transformed successfully?

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[Mega [Andreas Stuermer]]

 Plasmid rescue: Probably not 

"The DNA has resistance to spec and was an integration vector into amyE"

I wonder, integration should be a rare event, or shouldn't it be? 

[Koeng]

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.

[Cathal Garvey]

> 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?

[Koeng]

Ya, i didn't find the mutant, but I did originally plan out for using it for Bacillus minipreps. The second I saw "lysozyme" I thought "lysis genes" and tried to find some inducible lysis genes, because I am super lazy and don't want to use lysozyme.

On the Bacillus recombination system, it is probably the most regulated biological system I have ever seen/worked with. They have entire reviews on *The uptake, *the recombination, and the *regulation of expression of these genes. 

Yep, the mitochondria mouse genome paper and "combining 2 genomes" used huge amounts of DNA (1mb) but still got successful transformations... plus in one of the papers on competence it says that DNA coming into the cell gets sheared but in both of the first 2 papers they were still were able to transform. Without recombining into the genome, it should be even easier.

-Koeng

[Koeng]

Good and bad news

Bad news- The transformation I attempted yesterday to get titers didn't work. Like at all. However I did 2 things differently

1) I added media the first time, yesterday I just added additional xylose

2) The first time I allowed the growth time to be near 1 and a half hours, this time i just gave em an hour

Good news- Now i know things to optimize.

Next week I'll continue experimenting

[Koeng]

I mean this time I gave them ***half a hour