I need to take say 50kb gene cluster from the chromosome of one bacterial species and transfer it to another species. The gene cluster should be expressed. I want to observe if (how) the phenotype of the recipient species will change.
Literature?
Experience?
The classic method is to use an Hfr plasmid, coupled with selection for recombinants. Basically an Hfr is a "fertility" plasmid that facilitates conjugal chromosome transfer between bacteria. When the fertility plasmid incorporates into the chromosome it actually tries to transfer the entire chromosome from one cell to another. If you put an Hfr into the chromosome of your donor species near the cluster of interest, you could in principle conjugally deliver the entire cluster (plus lots of other DNA) to the donor species. Back in the 70's Postgate and Dixon used this method to transfer the nitrogenase operon (30KB) from Klebsiella to E. coli. For this to work, though, the donor cluster has to be able to recombine with the recipient's chromosome somewhere, and you need to have a good selection method to find the vanishingly rare cells that have incorporated the cluster.
You can also look for cosmid/fosmid cloning systems. I used them to make gene libraries but they can also be used for expression. The problem is you have to select your right piece of DNA to clone either cutting out of gel or by some other means.
Hi,
You could try the Method designed by Morimoto et al 2011: Methods Mol Biol. 2011;765:345-58. It employs double crossover though. So might need to make your synthetic cassette by PCR. This way you could include a homologous region of the host genome. E.g. in the amyE locus of bacillus.
cheers,
TS.
The easiest way would be to have restriction sites in both extremes of the cluster, cut it and ligate into a vector, nd then introduce it in your host. but that would be very lucky. In case you have not, you can make PCR of the cluster by parts (so you avoid the high mutatin rate after several kb), inserting restriction sites in each of the primers to ligate the fragments afterwards. 10 PCR would work. Take into account to keep the reading frame of the cluster after introducing your restriction sites.
As terrens Saaki suggests, doulbe crossover would work as well, but it is more difficult that simple restriction and ligation, i would say
Good luck.
Please, refer to intergeneric protoplast fusion technique published using Cellulomonas and Bacillus, wherein multigenic phenotype encoding genomic fragments are exchanged. It will be necessary to have specific selection marker(s) for each bacterium so that desired recipient bacterium could be selected. a review has been published in Biotechnology Techniques as well.
Brian Kvitko
donor species is Streptomyces. im not sure about an acceptor yet
I am working currently with Streptomyces. Take care of codon optimization, in case you use E. coli or another receptor with lower GC content. Also, you should remember to use DMSO between 3 - 10% for PCR, in case you use it.
May i know which Streptomyces specie are you using?
Carlos López
this is just possible plan for the future. Im new in this field of research (Actinomycetes). When looking into genomes in genbank I noticed that behavior of streptomycetes might be coupled with some genes they encode. I choose some clusters that might be responsible for phenotype and "social life" of Streptomycetes. So the goal is to tranasfer those genes and make other bacterium behave like Streptomyces do (organized colonies ....). maybe a crazy idea, but why not. The more crazy experiment, the better impact factor (...if it works). Anything gram negative is wrong i think. Acceptor must be species with sequenced genome, with similar codon usage and must be gram positive.
Perhaps this can work for you
Appl Environ Microbiol. 2013 Jul;79(14):4440-7. doi: 10.1128/AEM.00711-13. Epub 2013 May 17.
A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element.
Miyazaki R, van der Meer JR.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques