4 replies to this topic

[Fang Shi Tong]

We are trying to use Gene Bridges' Red/ET system to clone by homolgous recombination. We are able to introduce the rpsl/kanr cassette following selection for kanamycin resistance and streptomycin sensitivity. However, when we perform the second recombination step in which we should replace the rpsl/kanr cassette and recover strep resistance ONLY after recombination, we find that we still get plenty of streptomycin resistant colonies in the control (i.e. Red/ET induced with arabinose but mock electroporated.

Has anybody see and worked through this problem?

Edited by Fang Shi Tong, 21 April 2009 - 11:57 PM.

[phage434]

Many common lab strains are already strep resistant.  Even worse, mutation for strep resistance is common in E. coli in general.  Strep is likely a poor negative selection marker.  Make sure your strain is strep sensitive before you start.
Try a control experiment where you grow sensitive cells on a strep plate and check for mutant.

[Fang Shi Tong]

Thanks for the reply. The procedure makes use of the fact that the starting E. coli strain is "naturally" strep resistant. In the first recombinatino step, a wild type ribosomal gene (rpsl) is introduced to render the strain strep sensitive. In the second step, the rpsl gene is replaced with the insert of interest, and the bugs should now revert to strep resistance. As I said, we get too many. Interesting idea that rpsl might be mutating to strep resistance, but this should be a multi-copy plasmid, and according to GeneBridges, the presence of one wt rpsl gene should be enough to convert a strep resistant bug to strep sensitivity. We have checked for spontaneous conversion to strep-resistance (before trying to replace rpsl with our gene of interest) and it doesn't seem to be a problem.

[aquita10]

Fang Shi Tong, on Apr 22 2009, 09:56 AM, said:

We are trying to use Gene Bridges' Red/ET system to clone by homolgous recombination. We are able to introduce the rpsl/kanr cassette following selection for kanamycin resistance and streptomycin sensitivity. However, when we perform the second recombination step in which we should replace the rpsl/kanr cassette and recover strep resistance ONLY after recombination, we find that we still get plenty of streptomycin resistant colonies in the control (i.e. Red/ET induced with arabinose but mock electroporated.

Has anybody see and worked through this problem?

I thought the control should always be a non-induced control. Maybe you have some contamination...

[Fang Shi Tong]

aquita10, on Apr 30 2009, 03:13 PM, said:

I thought the control should always be a non-induced control. Maybe you have some contamination...

We've done both controls. Yeah, we were also worried about contamination, but we went to some trouble to eliminate(?) that problem.

Perhaps you could tell me the frequency of strep-resistant colonies that you get in your uninduced control (#colonies/ul bacteria plated).

Thanks.

Edited by Fang Shi Tong, 29 April 2009 - 11:24 PM.