4
$\begingroup$

I was looking at bacteriophages and how they're used to transform E.coli. While the whole process of how a bacteriophage works makes sense theoretically, I wanted to know how one goes about changing the default payload of a bacteriophage with the payload of interest (engineered plasmid) in practice?

Clarification:

payload - Genetic material that the phage inserts into the bacteria

default payload - Whatever genetic material the phage inserts into the bacteria naturally

payload of interest - Engineered plasmid that I want to insert into the bacteria

$\endgroup$
3
  • 2
    $\begingroup$ I would recommend looking at some methods sections. Takara has a lengthy guide for using different kinds of viral transduction methods in mammalian cells, which is a little different but quite similar overall, that may have some helpful low-level information. Here is a review that may be helpful, focusing on engineering methods for bacteriophage, with particular relevance to infectious disease applications. They go ove $\endgroup$ Mar 18 at 18:35
  • $\begingroup$ Can I ask why you want to use bacteriophage as opposed to electroporation or heat shock? I can't say I've ever heard of someone using phage to deliver DNA. I know I said it in my comment on your last post about electroporation, but it was more of a mental spitballing as to why lentivirus wouldn't work on E. Coli. $\endgroup$
    – yp66t89
    Mar 19 at 1:04
  • 1
    $\begingroup$ @yp66t89 I am actually familiar with some projects that were using phages to do rapid diagnostics type assays. I only made the connection once you mentioned retroviruses. But that got me curious about how you actually setup the phage payload in the first place. $\endgroup$ Mar 19 at 1:26
6
$\begingroup$

I am currently using a 'phage integrase' system to insert my gene of interest into P. putida and it is definitely a seamless process with high efficiency so I highly recommend it!

As for your question, the method through which we integrate the gene of interest using the phage integrase system is simpler than you think. We do not change the payload of a bacteriophage. We simply electroporate with two plasmids, one that expresses the BxB1 integrase and another plasmid that has our gene of interest that we want to integrate into the genome of P. putida. This integration is highly efficient, and it operates through a serine recombinase that recognizes specific attP and attB sequences in the plasmid that contains my gene of interest and native genome of my engineered strain. This method is significantly more efficient for engineering certain strains (typically non-model organisms) for genome integration purposes.

References for additional information on the system for your review:

Here is a paper that describes how we use the phage integrase system to effectively edit the genome. https://www.sciencedirect.com/science/article/pii/S2214030116300438

Here is the paper that discovered and characterized the mechanism this system operates through. https://www.sciencedirect.com/science/article/pii/S1046202310003142?via%3Dihub

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.