Recombinant work with in AT rich bacillus DNA in E. coli

Scott Coutts scott.coutts at med.monash.edu.au
Fri May 7 06:24:12 EST 2004


Trond Erik Vee Aune wrote:

> We have general problems with cloning and manipulations of Bacillus DNA 
> (AT-rich!) in E. coli (DH5 alpha), related to DNA instability, difficult 
> to clone, etc. We would be happy to know if anyone knows certain general 
> tricks (appropriate E. coli host, growth temperature, DNA preparation, 
> etc) that is useful when working with such DNA??
> 
> trygve

Yeah, I work with Brachyspira which also has a low G+C content (around 
24% in coding regions, as low as 7% in non-coding regions).

For expression work, try using the CodonPlus-RIL strain. Just do a 
search on the web for "CodonPlus RIL" and you'll find it. Also try 
looking up a couple of E. coli strains called "C41" and "C43". You can 
find them in the literature if you do a search. I don't have the 
reference on hand at the moment.

Most molecular biology techniques are the same, however. DNA preps are 
the same. PCR is usually troublesome, often requiring annealing 
temperatures down as low as 48oC, and careful optimisation of primers.

Cloning isnt usually too hard, but of course you need to pick your 
enzymes carefully because (aside from methylation) you are not going to 
be able to use a lot of them because they're recognition sites are too 
G+C rich.

If you're doing protein work, then the growth conditions you use will 
depend on the protein you're trying to express. It's not really G+C 
dependant.

Scott.




More information about the Microbio mailing list