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
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