Please help. 2. In class we explained how gene replacements can be performed in
ID: 34300 • Letter: P
Question
Please help.
2. In class we explained how gene replacements can be performed in species with high rates of homologous recombination, such as bacteria and many fungi. The example in class involved a gene knockout, but the same approach can be used to exchange one sequence for another. Suppose that someone gives you a 3 kb BamHl fragment of DNA that encodes a Pseudomonas sigma factor. Describe an experiment that you could perform to test if that gene could functionally replace the sigma 70 gene of E. coil.Explanation / Answer
To perform homolgous recombination, the sequence of sigma 70 gene of E.coli is found out. With this information, it is possible to replace any gene with a DNA construct of interest (BamH1 fragment of DNA that encodes Pseudomonas sigma factor). Next, the DNA construct (BamH1 fragment) is designed to include flanking DNA that is identical in the sequence of targeted gene. In addition to the positive selection marker- i.e.antibiotic resistance is added to the replacement vector.
The engineered construct is then added to cells which contain the targeted gene of interest.During meiosis and mitosis, the homolgous chromosomes align and the engineered construct finds the targeted gene and recombination takes place within the homolgous sequences. This phenomenon results in new peice of DNA fragment inserted into the gene of E.coli .
The rest of the gene will be unaltered but the single targeted locus will be replaced with the designed construct and some of its flanking DNA. Finally, the cells that have undergone homologous recombination can be selected by addition of antibiotic to the growth medium (positive selection) in which cells are cultured.