9 replies to this topic

[dreamerbac]

Hi
I'm wondering if I want to make a mutant in Bacteroides uisng SOE PCR. Would 800-1000bp fragment on each flanking side be enough for an efficient homologous recombination, like E.coli? Or it has to be around 2000-3000bp?
Thanks!!
  

Edited by dreamerbac, 12 November 2009 - 02:10 PM.

[HomeBrew]

dreamerbac, on Nov 12 2009, 05:09 PM, said:

Hi
I'm wondering if I want to make a mutant in Bacteroides uisng SOE PCR. Would 800-1000bp fragment on each flanking side be enough for an efficient homologous recombination, like E.coli? Or it has to be around 2000-3000bp?
Thanks!!
  

We usually allow for between 1.0 and 2.0 kb flanks, and adjust the size (a bit larger on the low GC side) if one flank is different than the other in terms of GC content.

[dreamerbac]

Thanks! Is there a reason why you need a longer fragment for recombination?  Or is this just tradition?

HomeBrew, on Nov 12 2009, 05:49 PM, said:

dreamerbac, on Nov 12 2009, 05:09 PM, said:

Hi
I'm wondering if I want to make a mutant in Bacteroides uisng SOE PCR. Would 800-1000bp fragment on each flanking side be enough for an efficient homologous recombination, like E.coli? Or it has to be around 2000-3000bp?
Thanks!!
  

We usually allow for between 1.0 and 2.0 kb flanks, and adjust the size (a bit larger on the low GC side) if one flank is different than the other in terms of GC content.

[HomeBrew]

dreamerbac, on Nov 13 2009, 11:49 AM, said:

Thanks! Is there a reason why you need a longer fragment for recombination?  Or is this just tradition?

Way back, we found that we couldn't get plasmid integration via homologous recombination to work when using smaller flanks.  It wasn't an exhaustive survey, and might be strain-dependent  (we are mostly working with NCTC 9343) or even experiment-dependent, but we've always stuck with it.

[dreamerbac]

Thanks so much! I also wonder what is your tolerable GC% difference, as well as size difference, between the up and down fragments, ? would 4% difference need to be compensated? if yes, how much size difference can do?

HomeBrew, on Nov 13 2009, 10:58 PM, said:

dreamerbac, on Nov 13 2009, 11:49 AM, said:

Thanks! Is there a reason why you need a longer fragment for recombination?  Or is this just tradition?

Way back, we found that we couldn't get plasmid integration via homologous recombination to work when using smaller flanks.  It wasn't an exhaustive survey, and might be strain-dependent  (we are mostly working with NCTC 9343) or even experiment-dependent, but we've always stuck with it.

[HomeBrew]

The GC average, of course, doesn't take into account local variation, but we've found the high average GC flank crosses in (and out) much more readily.  This becomes an issue when trying to do allelic replacement, as you need the plasmid to cross out via the flank opposite to that used to cross in, or you wind up with only wild-type revertants.  So, to balance the cross-in/cross-out favorability a bit, we try to balance the average GC of the flanks by shortening the high GC flank and/or lengthening the low GC flank until they're as close as we can get them in terms of average GC.  Usually, we do this in increments of 200 - 500 bases, until we find a pair of flanks that match up as best we can, trying not to drop below 1 kb for either flank.

If the GC content difference is still around 3% or greater, we'll add a bit more (200 - 300 bp) to the low GC flank.  We do most of our flank generation by PCR, so the final flank size wiggle is dictated by the primer design -- we use Primer3 to design primers, and take the best set possible based on our desired flank sizes.

This is really more of an art than a science, but by following these design guidelines, we've had pretty good success in such experiments, given the dearth of genetic tools available for Bacteroides.

[dreamerbac]

This is very helpful! Also since the GC content is pretty low in Bacteroides, how long is your typical primer? Do you use longer than 20bp primers?

[HomeBrew]

Our typical primer is 24 bp (I set primer3 to minimum of 20, optimum of 24, maximum of 30) with a Tm of 60C.

[dreamerbac]

Thanks so much, HomeBrew! I really appreciate it!

[HomeBrew]

Glad to help, dreamerbac.  Good luck!