Sigma factor... Promoter in halobacteria

[Koeng]

Hello everyone!

Currently I am creating a halobacteria vector, and the (hopefully) final version is mutagenesizing in my PCR machine right now.

I have the origin of replication and everything in it, but the gene for chloramphenicol resistance is kinda problematic. I am mutagenesizing an RBS downstream from the gene, but how about the promoter? I am currently using the one from pBeloBac11, common always on promoter.... But how will it work in Halobacteria? Any guesses? Because if it does need a different promoter that is going to be a pain...

Koeng

I will also update when the results come in!

[Patrik D'haeseleer]

One thing to keep in mind is that Halobacteria are - annoyingly - not Bacteria. I think Sulfolobus is probably the Archaea that has the best genetic tools available. There should be some known promoters from there that you could use. It's an entirely different phylum than Halobacteria, but at least it's in the same Kingdom!

Another approach might be to use a promoter from a housekeeping gene in the Halobacteria you're targeting. If you have any transcriptomics data available, you could use that to pick a highly expressed gene. Alternatively, there are also some halobacteria phages that you might be able to mine for strong promoters. Phages and viruses often have stronger promoters than their host organism.

Patrik