I am very new to the concept of gene cloning and transformation. I am trying transfer the arenite oxidase gene cluster (9kb in length) to E. coli strain C600. I want the E. coli strain to express this gene in order to oxidize arenite for bioremediation of groundwater. I am just realizing how complicated it really is to select the right expression vector. I have not studies this topic at all at school (still in high school); I am just studying this topic on my own. I am worried that my 9kb sequence could be too big for the plasmid vector or the bacterium. I have been trying to find the right plasmid expression vector for days, and it seems like everyone I find isn't quite right.

I have been kind of vague, so here is the whole story: I am in possession of a gram-negative bacteria that can oxidize arsenite into arsenite very efficiently. The gene cluster that is responsible for this is Aro, which is 9 kilobases in length, and I am looking to use a heat shock method to transfer that gene cluster into a specific bacterium (I found that E. coli C600 might be a worthwhile candidate). I have everything I need for the extraction of the DNA and PCR, but I am just trying to find a plasmid expression vector that I can insert into E. coli C600. Originally, I looked at pUC19, but then I found out it is only a cloning vector and not an expression vector. Then I looked at the pET system, but there are so many selections; I cannot seem to find the right one.

Are there any suggestions for what vector I should use or any guidance on how to find one?

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    $\begingroup$ You can use an arabinose inducible promoter for expressing the gene(s) of your interest. There are several vectors that have this system. Search for "pBAD" expression system. PET system is generally used for protein purification. You perhaps don't want that level of overexpression. It's difficult to comment which vector would be the best without going into the details of your project. Note that expressing an entire pathway may not be that straightforward. Are the genes in this 9Kb cluster parts of the same operon? If not they will have their individual promoters. $\endgroup$
    Commented Feb 3, 2019 at 12:06


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