Hi,
I am working on a pathogenic streptococci strain harboring a ~20 kb sized plasmid.
I would like to generate a mutant by removing this plasmid from bacteria or inactivate its expression using any genetic engineering technic.
If anyone has experience on this or read well-described research paper, please give me comments. Additionally, is allelic exchange using homologous recombination (using suicide vector) still applicable on genes located in plasmid?
Please give me any suggestions and answers.
Regards
Allelic exchange will normally still work on a plasmid but be aware that if the plasmid is present in multiple copies you may not efficiently be able to target all copies. So some sort of post-segregation analysis will be needed to ensure you have a population carrying only your "mutant" plasmid.
Plasmid curing is more difficult, in E. coli the classical methods were to grow for a number of generations in acridine orange and then test for curing. But you need a phenotype for the plasmid to do so effectively.
Allelic exchange will normally still work on a plasmid but be aware that if the plasmid is present in multiple copies you may not efficiently be able to target all copies. So some sort of post-segregation analysis will be needed to ensure you have a population carrying only your "mutant" plasmid.
Plasmid curing is more difficult, in E. coli the classical methods were to grow for a number of generations in acridine orange and then test for curing. But you need a phenotype for the plasmid to do so effectively.
There are many methods to plasmid removal from bacteria such as using medicinal plant extract, metals nanoparticles, paraprobiotics, and antibiotics.
All methods of lysing bacteria will yield plasmid solutions contaminated with chromosomal DNA and RNA. Centrifugation removes the vast majority of chromosomal DNA (it will form a pellet, while plasmid DNA remains soluble), and treatment with RNase will eliminate contaminating RNA.
https://www.mybiosource.com/learn/testing-procedures/plasmid-isolation
There are two methods which are used for obtaining the plasmid DNA from the bacteria. They are named as alkaline lysis method and boiling lysis method. They both are having different working principles.
https://www.sanfoundry.com/genetic-engineering-questions-answers-purification-plasmid-dna/
Michael J. Benedik
Thank you very much for your reply.
I would welcome to try with your suggestion: bacterial culture for lots of generations with supplement of acridine orange or other suitable dyes.
One thing is that,
Is bacterial chromosome randomly get mutated after exposure to those media? I would like to generate a plasmid-cured strain with intact chromosome.
You can do a literature search for plasmid curing to find some protocols. While both chemicals do increase the mutation rate somewhat, I don't think it will be too high that you need to worry.
I recently saw a paper that used Crispr/Cas to cleave a plasmid and thereby increase the frequency of plasmid loss, if you have easy access to that technology.
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