Hi all,
I am making deletion mutants in Burkholderia pseudomallei by allelic exchange. Basically, I clone the deleted fragments into a suicide plasmid with Kanamycin (Km) resistance and sacB counterselection.
I conjugate the plasmid into B. pseudomallei, and select for transformants on LB Km 1000 ug/ml plates.
To resolve the merodiploid isolates by sacB counter-selection, I have tried growing merodiploids in Yeast Tryptone (YT) agar with 15% Sucrose. Then, I plated the single colonies grown (on YT agar + 15% sucrose) on LB agar and the selected colonies were then screen again using LB Km 1000 ug/ml.
I get isolates which Kanamycin sensitive, however it resolved / revert back into wild type (WT) instead of mutants.
Any suggestions?
Please, any help is welcomed! Many thanks in advance.
It may be that the mutation confers a negative growth phenotype, or it might possibly even be lethal. How many kanamycin-sensitive secondary crossover cfus did you screen?
Using Rhodobacter capsulatus, I and others have made several markerless mutants using a similar sacB-approach. As revealed by PCR screening, sometimes the ratio WT:mutant cfus wasequal, other times only one out of 50 had the correct mutation - the latter was generally for mutations that turned out to have a negative phenotype (altered/reduced growth).
Did you re-streak or cultivate your initial antibiotic resistant cfus (using antibiotic) before plating them on sucrose? If not, there is a chance that you had carryover WT-cells (which are not sensitive to sucrose). Also, in case you cultured your cfus in liquid culture with sucrose: if your deletion confers a negative phenotype, they may have been out-competed by the WT-revertants during this phase.
Due to the small size of the deletion, I think I would first design PCR primers that each side of the deleted region. 200bp difference should be easy to distinguish in an agarose gel of the PCR products, and I would think also the 60bp difference if using a high percentage gel (if not, try PAGE). Then, for cfus that only contain the smaller "deletion" band, I would re-screen by PCR amplifying the entire manipulated segment (ie. using primers that bind outside of the segment(s) that was included in your vector) and send it for sequencing.
For a gene that is presumed to be "difficult" to delete, an alternative approach could be to first make a marked mutant, and then swap this genetic segment for a markerless-deletion using the sacB system. That way, the first step that removes the function of the gene can be directly selected for.
Dear Alexander,
Thanks for the useful information provided!
I screened a total of 100 colonies for the 200 bp resolved merodiploids and 55 colonies for the 60 bp deleted resolved merodiploids.
I did re-streak my initial antibiotic resistant cfus (using antibiotic) on LB with Km 1000 ug/ml plates before plating them on sucrose.
I totally agreed with you that to design PCR primers that each side of the deleted region to validate the the resolved merodiploids because I did the same thing as you mentioned.
By the way, may I know how much bp did you knockout in your Rhodobacter capsulatus's experiments? Because I did read some comments before that saying that the SacB counterselection was best to knockout the gene size between 500 - 1000 bp. So, I am suspecting that the second event of homologous recombination cannot occur in my case due to the small fragment (that caused the formation of excision loop with target genes impossible) I tried to knockout. Anyway, is it possible?
It's not the deleted segment that needs to be 500-1000 bp, but the sites for recombination aka. your flanking regions (the "excision loop" will include the vector, which is typically several kb.)
So, your "upstream" and "downstream" sequence that you cloned into the suicide vector should each typically be >500 bp to allow efficient (RecA-mediated) recombination in your bacterium - and of about equal length to avoid higher chance of recombination on one "side" of your deletion than the other.
The smallest deletion I made using a sacB approach was about 160 bp (delta-ghsA).
I don't remember that this caused any specific issues.
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