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

Mostly the promoter regions. They are the area of widest divergence
between bacteria and eukaryotes, and the machinery needed to read one is
basically absent from the other.

So, to get a bacterial gene working in eukaryotes the *traditional way*
(that is, without using synthetic DNA technology, which is almost always
a better idea these days):
1: Isolate bacterial coding sequence. Either:
1A: Reverse-transcribe a pool of bacterial mRNA to get cDNA, then PCR
for desired coding sequence.
1B: Cut out desired gene with nearby restriction sites, purify, and then
snip as closely to the coding sequence as possible.
1C: Use PCR to amplify just the desired CDS (generally the best way,
because it's only one-step.)
2: Ligate coding sequence (CDS) to a suitable promoter+Kozak RBS and
terminator for the target species. Rho-independent Terminators can be
general-purpose and often work in many species, because their mechanism
of action depends on their DNA/mRNA sequence, not protein factors.
Promoters are pretty species-specific, and Kozak sequences (the ribosome
binding sites for Eukaryotes) can be somewhat species, tissue and
context-specific, also.
3: Ligate or PCR-concatenate on adaptor sequences that enable insertion
of DNA into a target region/site, either through integrases or
homologous recombination or just random insertion.
4: Proceed to transformation. Hope you don't have to codon-optimise,
which involves a PCR cycle for each codon you'd like to change.

These days, instead you would just do:
1: Download protein sequence for desired protein from Uniprot.
2: Back-translate to desired species' codon-optimised format (although
methods for how best to do this differ, and resources aren't available
for most species using the best current methods).
3: Paste in a suitable promoter/RBS and terminator, plus any desired
sequencing primer-binding-sites, cloning sites, integrase recognition
sites, etc.
4: Proceed to greater cloning strategy or transformation, depending on
how complete the project is by the time it arrives in the post.

You might assume that synbio is more expensive than traditional cloning,
but the time-saving factor alone means this is often untrue. If you know
your way around DNA to begin with, you'll save months of time using
SynBio for a medium-big project. For basic stuff like clipping GFP from
one plasmid to another, it's wasted money usually.

On 07/06/12 16:33, 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.
>


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