Dear scientists,
I have an enormous number of pseudomonas aeruginosa drug resistant strains, I would like to analyse the gene transfer ability of those bacteria through conjugation experiment.
As per literature, I understood it needs a recipient strain (which should be sensitive to common drugs) or other pigment marker.
Can anyone suggest a good recipient for pseudomonas, is it pseudomonas strain has to be used as recepient or E.coli or the other bacteria can be used as a recipient cell.
If any kit is available (can be purchased in India) pls do inform me.
Thanks in advance
I disagree with Jai Ghosh, though I would be interested to hear his reasoning.
You need a plasmid-free strain of P. aeruginosa. I think the laboratory strain PAO may well serve your purposes. You will need to select a mutant with a chromosomal resistance mutation. I would suggest nalidixic acid (100 ug/ml), rifampicin or azide; all of these are relatively straightforward to select: 100 ul of overnight culture on a selective plate will usually give enough colonies. Restreak to single colonies twice and make a frozen stock of your strain. It makes sense to pick three-or-four colonies in case your first choice turns out to be uncooperative. And do remember to check that your mutant is P. aeruginosa (microscopy, oxidase, odour, etc)! There are tragic tales of people who have carried out entire research projects on the wrong species.
As regards conjugation protocols, I would use the classic cross-streak mating procedure. On a plate that selects for the recipient resistance and one of the donor resistances you make a straight streak from a single recipient colony and streak donor colonies across it. Transconjugants grow down-streak of the cross. A rifampicin resistant recipient is preferable as rifampicin will not interfere with conjugation (nalidixic acid might).
This won't work all the time but it will alllow you to screen a large number of strains at the same time. The gold standard method is filter mating (or it was, when I was using it back in the 1980s), but it is rather laborious.
In the first step You have to use a plasmid free and antibiotic reasiant strain of the same genus of P.aeruginosa. Later you may try to realize your futher plans.
For conjugation, your recipient would ideally be a garden variety Pseudomonas strain, which is sensitive to a drug resistance marker whose transfer you are going to test. Therefore, there may/have to be different recipients depending on a particular conjugation. The tricky part is that for counterselection your recipient has to be resistant to a selective marker, to which your DONOR is sensitive. You can make your recipient resistant through transposon insertion, or by selecting for spontaneous resistance to e.g. rifampicin. It is difficult/impossible to suggest a recipient or conjugation strategy without knowing anything about your donor (s)
There are so many experiments done on conjugation process to confirm the resistance phenomena using E.coli K12 as a wild type.
E.coli K12 strain is a good option for initial conjugation experiments. if you have already profiled your isolates, select any two antibiotics; for one your isolate be resistant and for other it should be sensitive and this sensitivity/resistance should be opposite for recipient strain.
You may try E. coli K 12 Az Rif or E. coli K12 C1a Nal, or E. coli J53 Nx lac+, as well as E. coli W3110 Nx lac+, however the transfer from Pseudomanas is more difficult.
I disagree with Jai Ghosh, though I would be interested to hear his reasoning.
You need a plasmid-free strain of P. aeruginosa. I think the laboratory strain PAO may well serve your purposes. You will need to select a mutant with a chromosomal resistance mutation. I would suggest nalidixic acid (100 ug/ml), rifampicin or azide; all of these are relatively straightforward to select: 100 ul of overnight culture on a selective plate will usually give enough colonies. Restreak to single colonies twice and make a frozen stock of your strain. It makes sense to pick three-or-four colonies in case your first choice turns out to be uncooperative. And do remember to check that your mutant is P. aeruginosa (microscopy, oxidase, odour, etc)! There are tragic tales of people who have carried out entire research projects on the wrong species.
As regards conjugation protocols, I would use the classic cross-streak mating procedure. On a plate that selects for the recipient resistance and one of the donor resistances you make a straight streak from a single recipient colony and streak donor colonies across it. Transconjugants grow down-streak of the cross. A rifampicin resistant recipient is preferable as rifampicin will not interfere with conjugation (nalidixic acid might).
This won't work all the time but it will alllow you to screen a large number of strains at the same time. The gold standard method is filter mating (or it was, when I was using it back in the 1980s), but it is rather laborious.
Sometime apply complicated experiments, but a plasmid free bacteria can be used as a reecipient in the conjugation with a plasmid carrying strain. Other simple experiment to sew the plasmid profile in agarose gel ( CC , Oc and linear forms). A classic example Joshua Lederberg replica plating. The results are convincing.
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