Biochemistry Questions Biochemistry Questions / How can transposon insertion mutants be selected and identified?

Transposon insertion mutants can be selected and identified by using a temperature-sensitive plasmid to introduce the transposon on. To do this, clone the transposon into a temperature-sensitive plasmid, and then transform into bacteria. Grow the bacteria at a permissive temperature, allowing transposition to occur. By increasing the growth temperature to a non-permissive temperature, the plasmid containing the transposon will be removed and degraded by the host bacteria, leaving only copies of the transposon that have transposed into the host chromosome. By having a selectable marker in the transposon, such as antibiotic resistance or a fluorescence gene (GFP), mutant bacteria containing the transposon can be selected for. If antibiotic resistance is used, antibiotic-containing growth medium can be used to select for the desired bacteria. However, if fluorescence is used, fluorescence-aggregated cell sorting (FACS) can separate and sort bacteria based on their fluorescence.