E. coli chromossome integration

[Linden]

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!

[Koeng]

Check out the pOSIP collection on addgene. That's what I use for my E. coli integrations. Integrations using lambda red are both size limited and fairly inefficient

-Koeng

[Linden]

Thanks for reply, Koeng, but I don't have enough money for that!

I am still wondering where in the E. coli chromossome I could insert my gene, with no harm for the cell.

Thanks!

[Koeng]

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

[Nathan McCorkle]

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.

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-Nathan

[Koeng]

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

[Matt Lawes]

Always assumes that OP means integration and not homologous recombination. If the latter, throw it into the lacZ of the lac operon (in non deleted strains) ... And you even get a happy blue white screen with xgal.....

>matt

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[Nathan McCorkle]

On Wed, Nov 4, 2015 at 6:16 PM, Matt Lawes <ma...@insysx.com> wrote:
> Always assumes that OP means integration and not homologous recombination.
> If the latter, throw it into the lacZ of the lac operon (in non deleted
> strains) ... And you even get a happy blue white screen with xgal.....

Good idea. I would consider integration to be a superset of homologous
recombination... I hadn't considered other people seeing them as
separate things.

[Yuriy]

Why such scrutiny over placement of a gene? Do you have your genes flanked by homology with E. coli chromosome?
Some intergenic regions have quirky DNA. Maybe you could use that to your advantage.
If you are useing NHEJ, make sure you are working with a strain that's expressing gam homodimers, or bacterial Ku homo/heterodimers. That should make things easier even with liniarized DNA.
Alternatively there are intergrases you could use.

[Yuriy]

There is always zinc finger intergrases.

[Cathal Garvey (Mobile)]

Run a mile! ZFNs are dead technology for a reason. Only their murderers-by-patent, Sangamo, are still flogging that dead horse.

Even absent Sangamo's evilness they were never as easy to modularise as they first appeared. Loads of odd inter-module weirdness.

TALENs and CRISPR are much more promising.

On 5 November 2015 09:33:44 GMT-08:00, Yuriy <yuriy...@gmail.com> wrote:

There is always zinc finger intergrases.

--
Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Yuriy]

Who mentioned ZFNs? I only mentioned ZFRs. Why get twisted over patents? There is no mention of needing to commercialize this intergration. Is there?

[Koeng]

Zinc fingers, as Cathal said, are essentially dead. In E coli, they're simply unpragmatic because you need to go physically make one before you use it. I haven't seen any use of them as of late. When people can't use an sgRNA (when they do, for example, mitochondrial modification) TALENs are far superior. 

Homologous recombination, which uses lambda red, isn't the same as integration, which uses lambda integrase (yea it uses some homology, but is a different mechanism). When I say integration, by the way, I mean integration into the genome using a integrase because technically both are integration into the genome. Homologous recombination, using lambda red, will only transform your DNA into a defined location using overlaps.The integrase only integrates into a *specific site* and is far more efficient for normal strains. I have never heard of a zinc finger integrase, I'm not quite sure they exist. In fact, one of the objectives of some directed evolution projects (PACE) is to get new specificity for these integrases to use in human cells, which would be great for gene therapy. 

E coli has very little "free space" in their intergenetic regions, bacteria in general are fairly compressed. The only genes they really don't need are prophages or transposons which are just invasive elements.

E coli, without lambda red, won't homologously integrate things into their genome (efficiently). In order to do that, you must first transform lambda red (pKD46), then create competent cells of that strain and transform your linearized DNA, then select for the integration, then cure pKD46 from the strain. Sometimes the selection is CRISPR, sometimes its just KanR, sometimes it's just selecting against something that's already integrated. It can even be zinc fingers, if you wish. CRISPR is getting pretty popular lately, the lab I work at has begun to use it for integrations even in yeast. I agree 100% with Matt though, lacZ would be a way better choice than a prophage if you're going into a strain that allows it.

With an integrase, you bypass the homologous recombination. This avoids the requirement for lambda red, which can be useful. All you would do there is transform and you're set (or remove your marker with FLP). In addition, it is not restricted by restricted by size ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847246/ ), so it's a good choice if your gene size is >2500 base pairs. 

-Koeng

[Cathal Garvey (Mobile)]

The modularity issues I mentioned aren't to do with the payload (nuclease vs. Integrase vs. Tr-factor), they relate to the actual binding efficacy of multi-finger ZFs.

ZFs are unfortunately context sensitive in uncomfortable ways. Sangamo *claimed* to have algorithms to resolve this, but everyone else ended up using massive a degeneracy strategies like phage display to artificially evolve functional multi-finger modules. It worked, but was labour dependent and required a kit that required onerous MTAs to acquire.

Source: a PhD I started years ago when ZFs were still cool that ultimately abandoned ZFs before falling off a cliff.

On 5 November 2015 11:57:58 GMT-08:00, Yuriy <yuriy...@gmail.com> wrote:

Who mentioned ZFNs? I only mentioned ZFRs. Why get twisted over patents? There is no mention of needing to commercialize this intergration. Is there?

[Yuriy]

I got you.