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

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.

On 07/06/12 19:35, Andreas Sturm wrote:
> 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 <cathalgarvey@gmail.com>
>
>> 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.
>>>
>>
>>
>> --
>> www.indiebiotech.com
>> twitter.com/onetruecathal
>> joindiaspora.com/u/cathalgarvey
>> PGP Public Key: http://bit.ly/CathalGKey
>>
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>

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