Bacterial species like E. coli are easy to transform using a variety of methods, including both chemical transformation and electroporation. Chemical transformation is especially nice to use as it can be done in high throughput. I'd like to start working with Mycobacterium smegmatis, though, and have only been able to find electroporation-based transformation methods like the one in the paper linked below. Can chemically-competent mycobacteria be prepared? If so, are they just very inefficient? Is it worth testing some E. coli-specific methods with mycobacteria?
http://www.sciencedirect.com/science/article/pii/S1046592807000253
Interesting question. I have not heard of any protocols to prepare chemically competent mycobacteria cells. Although it may not be impossible, I feel that preparing chemically competent mycobacteria would be unlikely. This is mainly because mycobacterial cell wall is thick and lipidic, and is very different from the E. coli cell wall. I would love to know if this becomes possible. One interesting thought would be that M. smegmatis tolerates quite high temperatures. So, it might be feasible to heat-shock at a much higher temperature than what is used for E. coli. Maybe try 60C or even higher... It might work, but again this is just a thought.
Interesting question. I have not heard of any protocols to prepare chemically competent mycobacteria cells. Although it may not be impossible, I feel that preparing chemically competent mycobacteria would be unlikely. This is mainly because mycobacterial cell wall is thick and lipidic, and is very different from the E. coli cell wall. I would love to know if this becomes possible. One interesting thought would be that M. smegmatis tolerates quite high temperatures. So, it might be feasible to heat-shock at a much higher temperature than what is used for E. coli. Maybe try 60C or even higher... It might work, but again this is just a thought.
I was thinking that you might use protocols similar to the ones used for streptomyces:
Protoplasing, transforming the protoplasts and plating on selective osmotically stabilizing media. My former boss Richard Baltz pioneered this technology. I don't know if I am on target but it can't hurt to look into it.
If you do decide to go the protoplasting route (if even possible) you will have to optimize all of the media, both liquid for protoplasting and solid for plating protoplasts and also for protoplast transformants. We used osmotically stabilizing soft agar overlays to actually plate the protoplasts onto the osmotically stabilizing agar. The temperature and humidity (by drying plates to varying degrees) of incubation for optimal numbers of protoplats regenerating on the media must also be determined. When transforming consider restriction enzyme systems within the organism. You can get around the resistance by laundering the plasmid DNA through progressively more restrictive strains, unless the organism that you are using is less defensive with restriction enzymes for destroying foreign DNA and then you may be off the hook. If the strain that you do use restricts the transforming DNA you may have to use high concentrations of plasmid DNA to get transformant(s). Once you get one transformant the DNA isolated from that isolate is protected from restriction and can be used to transform other varients of the bacteria. Sterile PEG in varying percentages has been shown to be useful in some species of streptomyces that are recalcitrant to transformation. Maybe this is obvious, or maybe this is helpful?
"Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria"(http://textbookofbacteriology.net/tuberculosis.html). Nocardia orientalis reclassified as Amycolatopsis orientalis is transformable. Perhaps the organism Mycobacterium smegmatis is similar enough in nature to these bacteria that they can be turned into protoplasts.
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