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Homologous Recombination of 2 genes with greatly different lenghts?.


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#1 Chris22

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Posted 25 April 2012 - 09:43 AM

Hi

I have a query regarding Homologous recombination which I am struggling to find an answer to, or how to even ask the question appropriately. Apologies for the chicken english.

I am using a vector system to insert a gene within a chromosome of a G+ bacteria using HR "switch out" of a ThyA gene (on the chromosome) and insertion of my recombinant gene (on the vector with upstream and downstream fragments from the ThyA locus SOE'd to the new gene at 5' and 3' end).

This will hopefully create a ThyA auxotroph unable to survive without supplemented Thymine,and expressing my recombinant protein. (steidlar et al)

The issue I am wondering about is if the ThyA gene is approx 1000bp, and the recombinant gene is approx 3500bp long..........is it likely that a HR event will occur at all. Is there a limit to which the two fragments must be close to each other in size?

There may be a widely used term for this but I am not sure of how to put it, Uneven HR, Differential HR?

Any assistance or suggested reading would be appreciated. Or a simple "yes chris that will work perfectly!!"

Regards
Chris22

#2 bob1

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Posted 26 April 2012 - 01:42 PM

I don't think the size of the "insert" makes a difference, as this should form a loop style structure with the "arms" binding to the appropriate sites on the ThyA. The main issue will be to have long enough arms to ensure that this event takes place. I think about 500-700 bp should be enough.

#3 Chris22

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Posted 27 April 2012 - 01:25 AM

Hi Bob

Thank you for your reply. From previous publications, 1000bp arms were used to ensure the switch, however the insert and gene were very close in size.

I will design the vectors with these and see how it it goes, a little less doubtful now. Thank you for your advice.

chris




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