How do you knockout an E. coli gene without disrupting the rest of the gene cluster?

Question

I'm familiar with the method to build Keio collection using single-gene deletion with an antibiotic cassette. However, what happens when there are gene clusters or overlapping ORFs and you still only wish to knockout a single gene?

Answer 1

Let's say that we have gene A, followed by gene B so that they overlap with 100 bases. If you want to knock out only gene A, you can do the following:

• PCR gene A and clone it into a vector
• PCR gene B and clone it into another vector
• Insert a antibiotic resistance marker at the overlapping region of gene A and B
  • now you don't have functional gene A and gene B
• Transform your cells only with the vector that contains gene B

Now you have disrupted only gene A but you have a functional gene B.