Recently, I'm working on a bacteria isolated from soil. It's hard to transfom. I have used chemical transformation and electrotransformation methods. They all failed. Then I found phage transduction. But I found little articles which used phage as a tool to transfer plasmid. I want to know why? If I want to use phage as a tool to transfer plasmid, is it feasible? I think maybe the reason is it's hard to isolate a lysogenic phage.
Well, there are likely a few reasons. One is that most bacteria can be transformed via chemical or electrical methods. Another is biosafety. Using phage would be a different type of biological containment then bacteria alone. You would also need to find a phage that infects your bacterial strain.
Another option that may work for you is to try conjugation. If you can find a transformation amenable strain that can "mate" with your strain that's another method for plasmid transfer. For example, pseudomonas and Ecoli can conjugate.
Amy Klocko Amy, many thanks to your answer. I tried to perform conjugation too and also failed. I guess the reason is, this bacteria is a gram positive bacteria. Conjugation is more useful for gram negative bacteria. I don't know whether it's meaningful or not if I can explore a method for transformation by using phage as a delivery tool.
It is possible to use some lysogenic phages to transduce plasmids but the real answer is very complicated. First it will depend upon the phage, some phages such as lambda will only package their own DNA (or DNA with the cos site), so you can package a cosmid for example but not a typical plasmid. Also many (most?) phage package specific amounts of DNA that fit the phage capsid. For example P1 phage packages around 90kb and is not going to effectively carry a small plasmid, or one much larger than that.
So there may be packing restrictions based on site and there may be size restrictions. Having said that, there do exist phages that are more flexible. But you would need to know the underlying biology of the phage to know. Or if you have good selections you can try and see if it works or not.
Regarding conjugation, again your plasmid will have to have the appropriate transfer signals for the conjugative apparatus to recognize and transfer the DNA. If you are working with large natural plasmids they might carry their own system, but if these are laboratory engineered plasmids then you would need to look whether it has the right sequences or not.
Also, do you know for certain that the plasmid you are attempting to introduce into the new strain is compatible and will replicate in that strain?
Having said all that, it might be worthwhile to work at optimizing your electroporation method, it might be just some minor technical problems.
Michael J. Benedik Michael, thanks for your answer. It's useful for me that I didn't know many phages only package specific amounts of DNA. It will be a limitation when I use phages as a tool to transfer my standard plasmid. And I extremely agree that, plasmid compatibility in a new strain is quite important to confirm. But, I can't verify whether a plasmid can work in a new strain without delivering. So, I think a suitable delivery method and a compatible plasmid will be verified at the same time. Otherwise, we will never know which one can't work. I will try to optimize electroporation later. Many thanks again.
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