[DIYbio] Re: Bacillus: An alternative for E coli in DIYbio?

Say you want to express a protein with a T7 or T5 promoter are you just going ask someone for the prophage and lysogenize these strains? Genetic editing is probably much more difficult without Warner strains and such. Do you know if Bacillus can produce comparable amount of protein compared to E. coli using T7/lac/IPTG? Does Bacillus restrict non-methylated DNA? Would make working from PCR/synthesized products difficult. Seems like an interesting hobby to test these things but Scientists probably will not use Bacillus just because it is slightly better than E. coli as E. coli has been used and tested and strains have been perfected for 10s/100s of years? Maybe Bacillus would have been the correct choice to start with cloning 100 years ago but  it is kind of too late. Remember you are fighting against years and years of research and optimization(probably more than a single lifetime) with the thousands/millions of strains of E. coli it doesn't seem like the _best_ choice. Though I am sure it could have some advantages maybe it would be good to focus on those.

Josiah

On Wednesday, January 1, 2014 5:16:45 PM UTC-6, Koeng wrote:

Hey guys!

Recently I have begun using Bacillus subtilis a lot, and I gotta say... it is a LOT easier then E coli. For example I did one experiment with a strain of Bacillus that over expressed comK under a xylose inducible promoter... all I did was get the cells in growth phase, give them some DNA, grow them for 45 minutes then plate. I got transformation efficiency of 6x 10^3 with a circular integration plasmid. That is even easier then Cathal's Bacillus transformation protocol (no offense (:  ), which is one of the simplest out there. I can easily get better expression of comK by optomizing the RBS and I can make this work under  a sucrose inducible promoter! (Working on that right now) In addition to this, I have found a way to negatively select in Bacillus (I think it is the mazF gene from e coli), meaning that I could possibly make a strain which all you need to do is transform them, no selection method needed! 

Plus Bacillus can take pure PCR fragments... meaning that using homologous recombination and negative selection you could literally PCR an interesting protein from lets say, some cheek cells, then integrate it into bacillus. The integration could then be expressed, secreted, and then purified (using a variety of tags already in the bacillus, given that the gene has no introns/exons) Skip plasmids and e coli entirely! (of course... I need to make these strains)

Anyone have anything to add or conflicting views?

-Koeng