Microbial gene knockout

[sahithim]

Hi, I'm looking to do a science project for which I need to examine the effects of a knockout bacterial strain.

I know exactly which gene I want to knock out (buk, which produces butyrate kinase) but I don't know how to go about doing it. From what I have heard so far, I would need to make a modified plasmid that would then be taken up by electroporation or heat shock, which I understand. However, I do not know how to disable buk in the plasmid.

Any help would be greatly appreciated!

[Ryang174mako]

you could do a knockdown that produces sirna or shrna (which knocks down the expression of the target gene) and overexpress it in the bacterial strain this would be done after electroporation or heat shock and that's it. it won't knock it out though it produces the same effects but a knockout is done by a deletion of the target gene and the repair the genome without the target gene.

[sahithim]

Thank you! Could you tell me more about how to perform a knockdown?

[Ryang174mako]

I only know what I've read I'm not an expert but it pretty much just shuts down the gene for a little bit, in some cases, to as long as the cell lives for others but the gene is still intact, now I think that the gene is still expressed but it's extremely low if at all but its as easy as any other gene modification but instead of choosing a plasmid with the promoter the specific genes cdna and all the other stuff your gonna choose the promoter and the specific genes sirna. if you do some Google searches you can learn more about gene knockouts and knockdown but knockouts are a little more complicated from what I can tell. I've never modified a gene network because I'm still in high school but I am interested to the point of filling my free time to research how it is done, it has to do with things like crispr and Cas 9 which are the easiest and simply choosing a specific gene or locus which is the part that confuses me.

[sahithim]

Yup, I definitely relate. Thank you though!