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Unformatted text preview: Lecture 13 Lecture 13 Lecture 13 Lecture 13 Lecture 13 Gene Manipulation in Bacteria Gene Manipulation in Bacteria Gene Manipulation in Bacteria Gene Manipulation in Bacteria Gene Manipulation in Bacteria There is no meiosis in bacteria so special techniques have been worked out for manipulating genes in bacteria so that mapping experiments, strain construction, and complementation tests can be done. First, we need a way of getting chromosomal DNA from one cell into another. There are several ways to do this. All of the methods have in common the use of special extra chromosomal elements for mobilizing chromosomal genes; the methods differ according to which extra chromosomal element is used. We will consider a method that uses phage and is known as Transduction Transduction Transduction Transduction Transduction E. coli chromosome is 4.6 x 10 6 base pairs Phage P1 P1 P1 P1 P1 chromosome is 10 5 base pairs After infection of E. coli , the phage DNA is replicated by a mechanism known as a rolling circle and the phage is packagedinto phage particles one headfull at a time: 1/300 phage mistakenly packages E. coli chromosome DNA instead of phage DNA. Each phage particle will package about 1/50 of the E. coli chromosome. By combining probabilities we see that about 1/15,000 phage will carry a particular E. coli gene. A basic transduction experiment to measure the linkage between markers A and B is done as follows: (1) Grow P1 on A + B + (2) Infect A B (3) Select for A + and then screen for B + The idea is that we are looking for the rare cases where some chromosomal DNA carrying gene A is moved into the recipient. To find these recombinants, we select for A +...
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