Re: [DIYbio] Re: Genome editing tools, gene deletion?

There are lots of easy ways to do genome editing in Bacteria mostly using homologous recombination. A common one is recombineering http://en.wikipedia.org/wiki/Recombineering . Another is through conjugation.

 

On Sunday, March 24, 2013 11:08:18 AM UTC-5, Koeng wrote:

Class 2 I believe? Fokl is a class 2, and the site it binds to can be engineered (TALENS or Zinc Finger). So hypothetically if I add a ligase to it, they will cut the dna then ligate it together? The problem I am thinking of us if I attach a ligase, the Fokl enzyme will cut then the ligase will ligate the stands back together! Would it be possible to ALSO attach a enzyme to blunt the sticky ends so then the 2 ends of the chromosome will be able to ligate together?

I checked out miRNA too :) it seems pretty cool, but the only problem is I am not looking for gene silencing, but entire gene deletion. I was thinking of perhaps using this to created a minimal bacterium by using a plasmid, each with synthetic TALENS to delete one gene. But that is just an idea :)

-Koeng

On Sunday, March 24, 2013 3:39:03 AM UTC-7, Cathal Garvey (Phone) wrote:

Splicer complexes like miRNA antisense systems are fairly exclusive to RNA, I think, although you could probably (if so, someone may have already) hack it otherwise.

To selectively excise a chunk of DNA while closing the ends neatly, you could use a integrase/excisionase system, but for the the flanking sequences and resulting 'scar' site must be predefined according to the enzyme.

There's a class of restriction enzyme that excises a chunk of target DNA,sometimes without requiring or leaving boundaries, but they don't repair the free ends. Fusing one to a ligase might help by ensuring that DNA only gets excised when a repair enzyme is close at hand.

Mega <masters...@gmail.com> wrote:

But gene deletion should be much easier with miRNA, if I'm correct there. It doesn't need a targeted integration, just any place is valid. 

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