Re: [DIYbio] Re: DNA design questions

it would work better than having your sequences homo recomb'd out....
also remember what Cathal said about too strong being bad in some
cases. certainly 1000 polymerase per second vs 900 is not much
different... i don't know what average polymerase per second flux is
for low, medium, strong, strongest promoters... does anyone actually
use polymerase per second??? (other than the igem folks or whoever
created the term)

is poloymerase per second (PoPS) actually a good way to judge a
promoter? It seems decent, but it doesn't address overexpression
leading to inclusion bodies, etc....

On Mon, Oct 29, 2012 at 2:32 PM, Andreas Sturm <masterstorm123@gmail.com> wrote:
> Other promoters won't be sufficient, because it have to be the strongest
> available. And there's only one strongest.
>
> The same promoter with different codon bias? Sounds great, but are you sure
> that works? I have never heard of that before. But it may be true.
>
>
>
>
> On Mon, Oct 29, 2012 at 10:19 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
>>
>> 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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--
-Nathan

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