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knock-out mutant through homologous recombination

suicide vector mutant homologous recombinatio

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#1 thefallengrace



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Posted 26 December 2013 - 09:52 AM

Hi there,


I am at the end of constructing my gene replacement vector.

The backbone vector is from pDM4, a suicide vector with Chloramphenicol as the marker and SecB as the counter-selection marker.


The gene replacement vector consists of a whole gene, which i want to delete but with an antibiotic resistance gene in the middle of the targeted gene. The targeted gene is around 1 KB while, the antibiotic resistance gene is 900 BP. 


I cloned the gene into a TA vector, and then cut in the middle of the gene with RE and insert the antibiotic resistance gene in the middle of the targeted gene.


Here is what it looks like:




Where, A is the targeted gene, and B is the antibiotic resistance gene. As you can see, the antibiotic resistance gene will disrupt the expression of the gene.


The problem is, as a rule of thumb in my lab, we need at least 300 BP as the upstream and downstream in order for homologous recombination to happen between the genome of the bacteria and the gene replacement vector.


However, due to some difficulties which cannot be avoided, i am left with one choice, which is to have around 200 bp as the upstream and around 700 bp as the downstream. 


Here is the question, will homologous recombination happen? i need some inputs from all experts in here, as i reached my dateline of my candidature, n i need this to happen. My supervisor and seniors, think it can happen, but it might be difficult to come by, hence need to screen more mutants, and some of my seniors said, it will stuck at meridiploid stage.



any suggestions or insights are greatly appreciated, thanks!




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