[DIYbio] Re: Bacterial gene expressed in eukaryonts and vice versa

- A frequent issue with recombinant gene expression is that your recombinant protein might not be able to fold properly in its new environment (whether that's due to different cytosolic conditions or lack of necessary chaperones, etc); if the protein doesn't fold properly, then it will aggregate into inclusion bodies, making it inactive.  A common example are highly disulfided proteins that are unable to fold in the E. coli cytoplasm due to the highly reduced environment, but it seems like you're interested in expressing bacterial proteins in eukaryotes, so you should have less difficulty (generally) with recombinant expression

- On synthesis - you can get 500-mer DNA fragments synthesized by IDT for $99 via their gBlocks line, which is incredibly cheap, plus they ship to you in 4-5 days (assuming that the sequence is reasonably easy to synthesize/doesn't have too many repeats/etc); if you already have a plasmid with a promoter/RBS/terminator, then you can get a small protein (or larger one, if you combine 2-3+ gBlocks) synthesized in that gBlock and then digest/ligate it into the plasmid for quite cheaply

On Thursday, June 7, 2012 8:33:46 AM UTC-7, Mega wrote:

It's me again :D

I was wondering what the problems were when introducing a bacterial gene into an eukaryont (easiest case: GFP or lux into yeast).


I identified some:

1) Codon usage:
When the codon bias is very different between them, it will give lower expression levels.
But it's not a show-stopper as the protein is still produced.

2) Ribosome binding Site:
According to this, it's not a big problem for bacteria->eukarotes.
http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/translation-systems/general-articles/ribosomal-binding-site-sequence-requirements.html

"Our data demonstrate that in contrast to the E. coli ribosome, which preferentially recognizes the Shine-Dalgarno sequence, eukaryotic ribosomes (such as those found in retic lysate) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites."

in the direction plant gene/mushroom gene/human gene -> bacteria this will be a problem.

Is my thinking  about RBS correct??



3) Promoter:
A bad promoter will give you bad results.