Hi,
I am struggling with my transformation experiments. I am studying bacterial strains that seem resistant to carbapenem antibiotics. What I did is the extraction of natural plasmids (because the resistance genes should be carried by them) to investigate if they are responsible for conferring the resistance when they are transformed to a competent e.coli strain. Each of my transformation reactions consisted of 50 ul of DH5a cells and 3ul of plasmidic extract. I did a heat-shock procedure and I selected on different antibiotics at different concentrations:
1ug/ul and 0.25 ug/ul meropenem (a carbapenem)
1ug/ul cefotaxime
1ug/ul ticarcillin
1ug/ul and 100 ug/ul Ampicilin
I did not have clones, except for few colonies on the 0.25 ug/ul meropenem and 1ug/ul Ampicillin. The gene I am studying is a little bit critical because it is known that it can trigger high levels of resistance when other resistance mechanisms are involved , for example defects in the membrane. But this gene, alone, could not be sufficient to give resistant strains. So, I had thought that this could be the reason of the lack of transformant clones on meropenem plates, although I am not able to see colonies on Ampicilin 100 ug/ul either (my positive control always worked in these experiments). Nevertheless, I did PCR on the few colonies I obtained on the 1ug/ul Ampicillin and 0.25 ug/ul meropenem for the resistance gene that the plasmid is supposed to carry. Indeed, I thought that at least for these colonies I had to find the plasmid, if the transformation worked and the DNA acquired by the e.coli clones. Does it make sense for you? I did not find anything either . So, does it mean that I don't have the plasmid at all in my transformant clones?
Then I tried to check if the strains , from which I extracted the plasmids are still resistant (I had tested it with MIC few months ago and they were resistant with a MIC at 64 ug/ul). I streaked the strains directly form the freezer stock to 16ug/ul and 32 ug/ul meropenem and I didn't have growth. But I was wondering if it is better to grow first these strains in a free-antibiotic medium and then streak them on meropenem plates (what I normally do for the MIC actually). Could the lack of growth depend on the fact the bacteria are streaked immediately on a “stressful” condition and they have not time to express their resistance?Or do you think I can just conclude they are not resistant anymore?
Any suggestion about these results would be really appreciated.
Thank you so much
1. What is your evidence for the resistance gene being plasmid born? Could it be a genomic resistance mutant? If it is genomic it is possible to make a genomic library from the resistant clone and transform a sensitive clones of a similar species (see below) with the resulting library and select on your anibiotic of interest.
2. If it is plasmid born, are you sure that the resistance gene will be expressed in E.coli? If it isn't then you might need to switch to a host species that can express the gene.
3. Yes you should probably grow your strains in antibiotic free medium. If I was doing this I would grow a small (2ml) culture, spread the entire culture onto antibiotic containing plates, pick resistant colonies, make overnight cultures and retest for resistance, freeze down single colonies of verified resistant clones. This might be overkill as I'm sure you followed a similar procedure prior to making you current frozen stocks, but I get paranoid.
It seems like your transfection procedure is fine so I would start with number 3 will investigating 1 and 2
Hi James,
thank you very much for your reply. I am not sure that this resistance gene will be expressed in E.coli. How can I do to know that?
Thanks
If the resistance gene is carried by a plasmid, it does not necessary mean that this plasmid will replicate in E. coli. Do you have any idea about the plasmid's nature? Have you sequenced it? From which bacterial specie does it come?
Maybe you can try to remove the plasmid from your strain (example: http://www.protocol-online.org/biology-forums/posts/39614.html) and see if it's still antibiotic-resistant (compared to the same strain with the plasmid)? Then you'll be sure that the resistance is carried by the plasmid.
Then, when you test your strain from the glycerol stock, it's better to let them recover from the freezing by growing them on a rich, antibiotic free medium. Then you can streak them on antibiotic-containing plates + antibiotic-free plates as a control. This is important because you can't know that the absence of growth means antiobiotic sensitivity if you don't check that the cells grow normally without antibiotics.
Good luck :)
Virginie makes a good point about plasmid replication in E. coli also. I'm not entirely sure how to answer your question, I saw your other question also so I will try and address both here. I it was me I would try the following:
1. Instead of using E. coli as a recipient, use a Klebsiella pneumoniae clone that is not resistant to the compounds you are studying.
2. Perform a plasmid extraction, transform the sensitive Klebsiella pneumoniae clone and select transformants on your compound of interest. If you get resistant colonies then it suggests your resistance gene is plasmid born. Bear in mind you will probably need to use a different transformation procedure than what you have for E. coli, and make sure you include a negative control to control for spontaneous resistance in the sensitive strain.
3. Once you are convinced that the resistance gene is plasmid born you could do one of two things:
a. directly sequence the plasmid you recover, if it is pure and abundant enough.
b. subclone into an E. coli expression vector with a selectable marker that gives no resistance to your compounds, select transformants on this antibiotic then test for resistance to your compounds of interest.
There are a number of ways you could do a and/or b.
Good luck, sounds like fun!
Thank you very much all of you.
I don't have at moment sensitive strains of Klebsiella. All of them are resistant and have plasmids.Is there a way to convert them to sensitive in lab? for example, plasmid curing could be a method ? How can I do it? Thanks
Quick question, your strains are resistant to antibiotics compared to what? E. coli?
If it plasmid born then it is possible to isolate a sensitive clone, as long as the resistance gene does not have an essential function in the normal growth of the bacteria.
To do this you would grow a liquid culture of your bacteria under conditions that do not provide selective pressure for the retention of the resistance gene, ie no antibiotic present. They will naturally shed the plasmid during replication. So you would spread your culture onto non-selective (no antibiotic) agar medium plates at a density that would give you about 200 colonies per plate (or maybe lower, you want single colonies that are well separated from each other). Once colonies have formed you would replica-plate onto two sets of agar plates, one set that contain the antibiotic, on that doesn't. If a clone has lost its plasmid then it will only grow on the antibiotic free plates and you can pick it from there. This is known as a negative selection screen and is done reasonably routinely with S. cerevisiae. You would use a velvet replicator pedestal like this: http://www.bio-world.com/productinfo/4_75_595/8468/Microbe-replica-plating-tool-mm.html.
Any cylindrer of the right diameter (mine was made with polysterene) and a rubber elastic band will do the job for plate replication ;) And you'll also need square pieces (about 12-15 cm) of sterile velvet. Works great!
And James is right, you really need a sensitive variant of your strain, as a control and to prove the resistance is carried by a plasmid. Otherwise you won't be able to say that your strain is resistant to antibiotics.
Good luck with your experiments!
- Badges
- Science method