Bacillus subtilis is not a "new species"... It is the second most studied bacterium in the world and has more information on its genetic structure then E coli. It has the most complete minimal gene set of any living organism
http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page
Bacillus subtilis may restrict some non-methylated DNA, but in literature it has been shown to be more effective to use PCR products then it is to use recombination vectors (given that they aren't linearized).
For protein production, if you wanted to express a protein with T7 or T5 promoter, just PCR the gene for each polymerase out of one of your e coli strains and replace, lets say, an integrated phage gene. Hell, you wouldn't even need to add a promoter or RBS, it would already be there if you do it correctly. Bacillus are also a lot more efficient at secreting enzymes then E coli, since they only have 1 membrane. Secreting the enzymes produced can be the difference between killing your cells with too high expression and keeping them alive
So rather, I would think that working with Bacillus is not fighting against but rather with years and years of research and optimization (tools for competence in Bacillus have been more explored then E coli, you can even "express competence" under a promoter with the protein comK)
On Friday, January 3, 2014 5:10:51 PM UTC-8, Josiah Zayner wrote:
It is great to try new things but the problem with starting with a completely new species of bacteria as a model genetic tool is that you are starting over or nearly as I am sure some research work has been done with Bacillus. I assume the strains of Bacillus are not endA- and recA-(meaning your DNA and the DNA of the host have a chance of being screwed up) and you probably cannot do blue white screening with them, a useful genetic tool.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
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