In article <Pine.PMDF.3.95.970123145756.139069C-100000 at oscar.allegheny.edu>, mcgeed at ALLEGHENY.EDU says:
>>>Does anyone know the minimum number of bp needed for a double crossover
>event at each end? Or an average range? I have a very small gene (200 bp
>on each end, disrupted with an antibiotic cassette) that
>won't transform. Of course the disruption could be lethal...
>Thanks
>David
>David J. McGee
>MCGEED at allegheny.edu Well, you didn't mention the organism, but being as you are in Rick Rest's
lab, I'll take a stab at gonococci. In the case of H. influenzae, I've
been told that 40-50 bp at each end should be sufficient to promote genetic
recombination. Why not take sequence flanking something like the opacity
protein, which is nonessential and can be monitored by colony opacity and try
to disrupt that gene? Put some sequence flanking the gene on either end of
an antibiotic resistance cartridge and transform it into piliated gonococci.
You could add different lengths of bp onto either end and monitor the efficiency
of recovery of your antibiotic resistance marker vs. flanking sequence length
and answer your own question. If your disruption is potentially lethal, you
could be getting recombination already with your 200 bp ("you may already be
a winner...").
Tom Dougherty
