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?
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.