Bacterial gene expressed in eukaryonts and vice versa

[Mega]

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.

[Cathal Garvey]

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.

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[Andreas Sturm]

Thanks, that was enlightening!!

Synbio sounds better and better :D

Do you have an idea/estimation on when synbio will be affordable? Will it ever be possible to print out a plasmid for say 100 $ ??
On the market everything is about demand. Will there ever be enough scientists/people to create that demand??

2012/6/7 Cathal Garvey <cathal...@gmail.com>

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[Cory Tobin]

> Do you have an idea/estimation on when synbio will be affordable? Will it
> ever be possible to print out a plasmid for say 100 $ ??
> On the market everything is about demand. Will there ever be enough
> scientists/people to create that demand??

Gene synthesis is around $US 0.30 per base pair right now. So if
your plasmid is only 333bp, then it's possible to make it for $100
today! Although, maybe not since most companies have a minimum order
of like $150 or so.

Here is a chart Rob Carlson published a while back showing the cost of
synthesis over time.
http://www.synthesis.cc/2008/11/gene-synthesis-cost-update.html


-cory

[Cathal Garvey]

I think there will come a point where DIY DNA synthesis becomes possible
and feasable. It'll probably suffer poorer quality and reliability than
the existing methods, but it will allow us to make our own DNA, provided
we're willing to accept a few errors.

That technology won't just be the beginning of "cheap" synthetic
biology. I think it's actually a critical piece of the future synbio
puzzle. The costs of failure are still too high in biology; we need
failure to be cheap or free!

So, hopefully within 5 years I'll have a DNA printer. But don't expect
them to be coming from lab equipment companies, and don't expect to see
many government grants going toward research into such equipment. I
imagine the notion scares the pants of biosafety committees who may be
consulted on such grant applications. No, I suspect (hope) it'll come
from us.

[Andreas Sturm]

You think it's possible within 5 years to build a DNA printer??
Or synthesizing will be cheaper??


I think as an exercise I'll look for the pVIB gene sequence and write it into plant DNA. (Codon bias).
Then I attach a promotor and eukaryont RBS ...(gene LUXA/LUXB/C/D/E)... terminator.
Thus correct?

[Andreas Sturm]

A question, Cathal... Is your plasmid high copy or low copy?
If it's high copy, the chance of getting lost is even smaller I assume.

2012/6/7 Andreas Sturm <masters...@gmail.com>

[Andreas Sturm]

Important question:

http://www.ncbi.nlm.nih.gov/nuccore/NC_006841.2?report=genbank&from=1044286&to=1045266&strand=true

This is the sequence of the LuxB gene.

Is there a promotor and terminator and RBS inside, or is this just the enzyme??


I'm hoping for the latter.
I'm translating it into plant DNA language. If there's the promotor, I have to find it and erase it, and replace it with a plant promoter. 

[Avery louie]

Heya Mega,

Good news!  The LUX operon has been successfully expressed in yeast.  Check out the post here which describes how it works.  Basically, it was cloned into two plasmids, and promoter has two genes downstream of it, with an IRES in between the genes.  An IRES is an internal ribosome entry site, so that the ribosome can transcribe the mRNA.

--A

On Fri, Jun 8, 2012 at 1:59 AM, Darren Zhu <darren...@gmail.com> wrote:

A couple additional comments:

- 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

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[Andreas Sturm]

Sounds terrific!!!!!!!

The difference between yeast and plants is very small compared to plant-bacteria.



Without restriction sites, it will be difficult to replace the promoter/terminator... Maybe possible with PCR

2012/6/8 Avery louie <inact...@gmail.com>

[Andreas Sturm]

Where can I get those awsome plasmids? :D

2012/6/8 Andreas Sturm <masters...@gmail.com>

[Avery louie]

No idea :(

--A

[Andreas Sturm]

So I still have to print my own code.

Multiple insertion will give you higher yields of the substance? Then  you could just print out the sequneces 2 or more times?

So why not make Lux AB CDE CDE??


It definitely will be expensive :(

2012/6/8 Avery louie <inact...@gmail.com>

[Andreas Sturm]

So I still have to print my own code.

Multiple insertion will give you higher yields of the substance? Then  you could just print out the sequneces 2 or more times?

So why not make Lux AB CDE CDE??


It definitely will be expensive :(

The one expressed in yeast was not codon-biased? It was just twice the background photons I read, so can you see it with your naked eye?

2012/6/8 Avery louie <inact...@gmail.com>

[Nathan McCorkle]

On Thu, Jun 7, 2012 at 3:08 PM, Cathal Garvey <cathal...@gmail.com> wrote:
> I think there will come a point where DIY DNA synthesis becomes possible
> and feasable. It'll probably suffer poorer quality and reliability than
> the existing methods, but it will allow us to make our own DNA, provided
> we're willing to accept a few errors.
>
> That technology won't just be the beginning of "cheap" synthetic
> biology. I think it's actually a critical piece of the future synbio
> puzzle. The costs of failure are still too high in biology; we need
> failure to be cheap or free!
>
> So, hopefully within 5 years I'll have a DNA printer. But don't expect
> them to be coming from lab equipment companies, and don't expect to see
> many government grants going toward research into such equipment. I
> imagine the notion scares the pants of biosafety committees who may be
> consulted on such grant applications. No, I suspect (hope) it'll come
> from us.

I agree that synthetic DNA will be on the desktop in 5 years, maybe
sooner. Having some error is actually desired in some situations such
as directed evolution, so early stage 'sloppy' techniques could be a
bridge to perfection that still allows for lots of biotech growth.

[Andreas Sturm]

5 years is far away from now.
The future shall be now! ;)