Stated simply, if you want to find sites that are negligible to growth, use 1) a site a current phage integrates in or 2) integrate over a defective phage which has already been there for a while or 3) consult the keio collection and find knockouts that do not influence growth.
-Koeng
On Wednesday, November 4, 2015 at 4:33:20 PM UTC-8, Nathan McCorkle wrote:
Koeng, I think the OP is asking how to find sites for insertion, where
that would cause negligible effects on the E.coli
health/performance/growth-rate/vitality. Getting it in there, whether
it's at a specific site, at some interspersed motif, random
insertion... are interesting, but slightly different questions. This
is somewhat a search problem, there may not be a site where specific
insertion doesn't cause any effects, but maybe there's some motif
insertion mechanism that usually produces no effects. Then you might
say, the exact site doesn't matter, it's the lack of effect that is
the goal for /whatever/ site ends up getting inserted into.
On Wed, Nov 4, 2015 at 3:51 PM, Koeng <koen...@gmail.com> wrote:
> A single pOSIP plasmid (https://www.addgene.org/45985/ ) will most likely
> save you 65$ worth of time, and plus you won't have to worry about size.
> What exactly do you want to integrate?
>
> If you still want to insert your gene using lambda red, put it in/on a
> defective prophage. Qin/Kim, e14, rac, CP4-57, CP4-6, DLP12 prophages would
> probably all work. (just looked quickly at E coli genome for these)
>
> -Koeng
>
> On Thursday, October 29, 2015 at 4:00:15 AM UTC-7, Linden wrote:
>>
>> Hey Guys!
>>
>> I would like to know how do I pick where in the E. coli chromossome I
>> should integrate my DNA so it doesn´t have any impact on other genes.
>>
>> Thank you for you time!
>
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