Construction of isogenic mutants help - Construction of isogenic mutants (Jul/21/2007 )

is there anyone can tell me or teach me how to knock out a bacterial gene please either by Transposon-based insertional mutagenesis or any other method. I have never done that and need to do it for me project.

or to the worse situation, are there any biotech companies do this type of service.

Transposon-based gene knock out is an inherently random process.

It is more of a case of looking for a transposase that works in your organism, flank a marker gene with the transposon elements, transform the cells, selected for colonies that integrate the marker. The do inverted PCR or similar techniques to find where your marker has landed.

Other methods that come to mind are homologoes recombination. Again you have your marker gene, this time it is flanked by sequence that is homologous to the region just flanking the gene that you want deleted. Linearise and transform in to desired cells, select for transformants, do colony PCR to find colonies where the gene of interest has been replaced by the marker gene. Additionally you could co transfrom plasmids in coding e coli recA and such to help the homologous recombination process.

I am unfamilar with companies, so can't help you there.

Basically it boils down to
-do you know the sequence in and around the gene you want to knock out. Is targetting an option?
-is the cell biology helpful, do you have a transposase for your organism, or how well does it homologous recombination work. How well does DNA transformation work?

Thank you.

What would be a marker gene in this case.
If you have any written protocol on how to do that, please send me.


In regard to transposon, someone suggested this kit for me (EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ Kit) from EPICENTRE.

http://www.epibio.com/item.asp?ID=286&...amp;SubCatID=46

Could please look at it and let me know what you think of it.

Thanks and I appreciate your help.

looks like a pretty standard transposase (Tn5) kit...
the marker in this kit is Kan resistence gene.
they have added an origin of replication, again pretty standrad.

The question is, will this transposase work in your organism? Does literature say anything in that regard? Has anyone sequenced (partial or complete) the genome of your organism?

Between the two, directed gene knock out by homologous recombination if far easier to do, then random knock outs. Just imagine the screening that a random knock out would require you to do.

I see your points.
homologous recombination then is my first choice.
Would you please direct me to a protocol on how to do it and where can I get marker gene?

firstly, what is the organism that you are using?
What do you know about its biology with regards to homologous recombination?
Has its genome been sequenced?

The marker gene, can be any selectable marker that is applicable to your organism. Antibiotic selection (if bacterial), antifungal (if fungus), antibiotics if you can do stem cell on your organism, GFP/YFP/RFP/CFP is you have to go via oocyte microinjection. (which may then actually require transposase to get gene knock out)

A google search will pull out more of the same
http://www.bio.davidson.edu/Courses/genomi...omolrecomb.html

I am working on bacteria "Brucella abortus" and its genome has been sequenced. some studies created some mutant strains, so I guess homologous recombination can be done.

There is a protocol on homologous recombination in current protocol in Molecular Biology that I found through the library. the gene markers cassette in this protocol can be amp, kana, cat. Some of them can be purchased from Stratagene and the strains used for the experiment can provided by some lab.

PLEASE SEE THE UPLOADED PDF FILE.

Thank you