Re: [DIYbio] Re: DNA design questions

Got a reference for the LuxAB only working once? That's really weird
sounding! Canonically, luciferases work as catalytic enzymes, have never
heard of them being exhausted so rapidly..

On 31/10/12 16:16, Andreas Sturm wrote:
> That's interesting. LuxG (or fre) was the limiting factor (in eucaryotes).
> Next it could be Decanal (CDE).
>
> Don't know if there is abundant luxAB. I read that this is not really an
> enzyme, because it reacts and is then junk (1 proper enzyme catalyses
> 10'000 molecules substrate, as you know) .
>
> So that would make Promoter1-LuxAB Kanamycin-Term- Prom2- LuxCDEG. So
> basically, there's not much diference in lenght.
>
>
>
>
>
> On Wed, Oct 31, 2012 at 4:36 PM, Cathal Garvey <cathalgarvey@gmail.com>wrote:
>
>> 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)
>>>>>
>>>>>
>>>>>
>>>>>
>>>>
>>>>
>>>> --
>>>> 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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>

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