Re: [DIYbio] Re: DNA design questions

homo recomb from what though? just use diff promoters and terminators
if that's what you were concerned about.... or the same promoter with
different codon usage

On Mon, Oct 29, 2012 at 1:00 PM, Andreas Sturm <masterstorm123@gmail.com> 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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--
-Nathan

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