I have a problem getting my sacB counter-selection to work. sacB sequence (obtained from PYUB870) was cloned into the MCS of pGEM-T, transformed into E. coli and plated on 1) 5% sucrose LB agar, and 2) 5% sucrose LB agar + ampicillin. Colonies grew on the ampicillin plate, while a bacterial lawn appeared on the LB agar + 5% sucrose. Why didn't the E. coli die by sacB?
If I'm understanding the experiment you transformed your plasmid and plated the transformation mix on the two media above? If correct then the proper experiment should have been to compare the # colonies on LB+Amp versus LB+Amp+sucrose. The reason you get plenty of colonies on LB sucrose is that any cells that lose the plasmid are no longer sucrose sensitive, so that experiment is pretty meaningless.
Hi Michael, thank you for your reply. That is exactly what I'm trying to do: To compare growth of E. Coli on plates with sucrose and sucrose+Amp, between transformed and untransformed cells. Unfortunately, while there is no dispute on the growth of untransformed cells (WT) on LB+sucrose media, I am wondering why will there even be colonies on LB+Amp+Sucrose. If the cells are untransformed, they will die by Amp. If they are transformed, they should die by sucrose. Why didn't they?
Two reasons why you see some colonies on Amp+Sucrose. First there can be some mutant plasmids that no longer express SacB. That is why the comparison of colony count on Amp and Amp+sucrose is important. Secondly the sucrose selection is not entirely effective. So the differential count will tell you what issue you are having. If the colony count on Amp is similar to Amp+Suc then you are having a sucrose issue and you might try a different E. coli strain. If you only get 1% or less the number of colonies then you are getting mutants or deletions or plasmid contaminants.
Hi, I am having similar issue in my experiments. As Michael explained above, there are several reasons like 1) sacB is no functional, or 2) selection medium is not good. In addition to the count check, proposed by Michael, what you can do is:
1 - pick your original plasmid, pcr amplify sacB gene, check sacB sequence. You see, I had a big surprise when I though the sacB gene located in a plasmid I got from my supervisor, was functional... but had 2 mutations in it...
2- Pick up successful colonies on LB-Amp, dilute in sterilized 10 miliQ water, strike 4 ul on LB-Amp and LB-Amp-Suc on the other side (make long line to see if any heterogenous colonies appears). Use LB-+Antibiotic+Sucrose at 6 to 10% rather than 5.
Hope the best for your experiment,
Arno
Hi Arno, thanks for your suggestions. You are right. When I tried using LB+10% sucrose, the selection was much better. Though there were still colonies on the plate, the numbers are lesser than when LB+5% sucrose was used. Indeed, like Michael mentioned, sucrose selection is not entirely selective.
Probably late, but, since this is archived it might be of use to others.
It is also common to eliminate salt from the medium, which apparently increases sacB activity.
Thank you Rahmi! That was very useful I was looking for why should I eliminate NaCl from my medium.
can somebody please explain, or maybe share some references, why eliminating NaCl from LB-sucrose10% increases sacB activity?
It is late, but it might be useful for someone else. Apart from the recommendations above, there is a key aspect that needs to be taken into account when applying any counter-selection protocol: the recovery phase after transformation should be long enough to allow proper chromosomal segregation. Bacteria carry several copies of replicating chromosomes during active growth. In that scenario, if only one of these copies still harbors the counter-selectable marker, that cell will be affected by it. By waiting until the cells are at stationary phase before applying the selection, one allows proper segregation to occur, and the population of cells will be composed by cells either with or without the counter-selectable marker, instead of killing most cells before marker segregation actually occurred.
I have a problem getting my sacB counter-selection to work. I want to select mutunt thought vector named pK18mobsacB(which contain sacB), I tried using LB+10% sucrose without NaCl or any other salt, but my sacB did not suicide, Is there any other method to allow sacB to die?@Roberto
Hi Fan-Yang Lv,
Could you please describe briefly your system and experimental conditions? (organism, reference for the protocol, etc.). That could give us an idea of where the problem might be.
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