Re: [DIYbio] Re: DNA design questions

I don't *think* that sequences around the length of a normal promoter in
such close proximity will be a big recombination hazard.. but it's
possible. One way to try to address this would be to read up as much as
possible on the promoter, and run BLAST on the promoter (accepting only
close relatives which are still active for comparison), and try to
identify which parts of the promoter are most important for function.
Then, make some changes to the apparently "unimportant" parts of the
promoter.

This is much harder in eukaryotes than bacteria, because promoter
structure is usually less "logical": in E.coli for example, the
consensus promoter for Sigma Factor 70 (the "constitutive")
transcription factor is:
TTGACANNNNNNNNNNNNNNNNNNNTATAATNNNN[CDS]

Whereas in Eukaryotes and Archaea you get common motifs such as the
TATAAA and the B-recognition sequence SSRCGCC* close to the CDS but with
less stringent spacing, and then you can have enhancers and such up to
several kilobases away. Regardless, this means in Eukaryotes, you have a
similar immediate promoter structure consisting of highly important and
unimportant DNA sequences, but unless you understand which is which you
can't as easily edit things to reduce similarity between otherwise
functionally identical promoters. At least, not without potentially
sacrificing promoter strength.

Something to consider: if I recall correctly, the limiting factor in
luminescence is luciferin availability. That is, having the same
promoter strength for LuxAB and LuxCDE should be a lower priority than
having LuxCDE under strong expression, assuming you can provide LuxCDE
with what it/they need to produce lots of Luciferin. Which is:
tetradecanal. Can't recall if LuxCDE is enough to convert Tetradecanol
to Tetradecanal, or would you need another enzyme to catalyse that..I
think not.

* that's IUPAC notation: S means "strong" = G or C, R means puRine = A or G

On 29/10/12 20:00, Andreas Sturm wrote:
> Sounds interesting thanks!!
>
> But if I do take Promoter -LuxAB - Trmnr - Promoter- LuxCDEG- Trn, the
> chance of homologous recombination wil be there.
>
> And I must take the strongest promoter to get any visible light ammount,
> I'd have to take it twice...
>
>
> If I include the viral 2a oligopeptide and *hope* it works, that will
> circumvent the problem with possible weak links?
>
>
>
>
>
> On Mon, Oct 29, 2012 at 6:22 PM, Cathal Garvey <cathalgarvey@gmail.com>wrote:
>
>> The longer the chain, the more chances there are for a weak link to
>> break it.
>>
>> Issues of transcriptional stalling or translational abortion/stalling
>> could end up killing the whole system, and it's hard to debug. At least
>> if you have several smaller fusions, you can try to identify which part
>> of the system is failing and fix just that.
>>
>> I'd suggest several smaller fusion proteins rather than one giant one.
>> Fuse the critical pairs or sets that form "bottlenecks" in the system:
>> LuxAB are cofactors, so fuse them. LuxCDE are steps in a synthetic
>> chain, so fuse them.
>>
>> On 28/10/12 16:50, Mega wrote:
>>> Does anyone by chance know how long an amino acid chain can be without
>>> getting problems with transcription and/or translation?
>>>
>>> Say you want to make a 6kbp long fusion protein (you clone the genes in
>>> frame and remove the stop codons) . That would make 2000 amino acids(!),
>>> and those sequences I've seen so far have around 400 amino acids.
>>>
>>>
>>> Will there be problems with the RNA polymerase or during translation?
>>> (Google didn't hit any adequate results, unfortunately)
>>>
>>>
>>>
>>>
>>
>>
>> --
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