We can also do a 3-factor cross to determine gene order. (1) Grow P1onA°B°c (2)Infect A-Bc (3 Select for A*and then screen for B+ and/or c+ Genotypes A+B+C+2 crossovers (A to C distance) A-B+C 2 crossovers (A to b distance) A°Bc 2 crossovers A+B-C+ 4 crossovers(very rare (Note that there are only four possible genotypes because we select at) A B A limitation of transduction experiments is the need for a good selectable marker. Tn5 insertions provide a way to extend the utility of transduction for mapping and strain construction For example let' s say that we have isolated a new mutation in the mota gene. MotA is a component of the bacterial flagellar moter and MotA- mutants are nonmotile, a phenotype easily detected by the inability of Mota- colonies to"swarm outward on soft agar plates. Imagine that we want to map the MotA" mutation or to move this mutation into an E. coli strain with a new genetic background. Clearly direct transduction MotA would not be possible since we have no way to select for rare (1715,000)transductants with the nonmotile MotA phenotype. One solution would be to use a nearby marker for which we can select to move Mota by its cotransduction with the selectable marker. Unfortunately, good selectable markers are not common and we are unlikely to have a good selectable marker placed within cotransduction distance of MotA- readily available. A powerful alternative approach would be to isolate a random Tn5 insertion that is close to MotA- and to use the Kanr trait conferred by Tn5 as the selectable marker for cotransductionWe can also do a 3-factor cross 3-factor cross to determine gene order. (1) Grow P1 on A+B+C+ (2) Infect A–B–C– (3) Select for A+ and then screen for B+ and/or C+ Genotypes A+B+C+ 2 crossovers (A to C distance) A+B+C– 2 crossovers (A to B distance) A+B-C- 2 crossovers A+B-C+ 4 crossovers (very rare) (Note that there are only four possible genotypes because we select A+) A - B- C￾A+ B+ C+ A limitation of transduction experiments is the need for a good selectable marker. Tn5 insertions provide a way to extend the utility of transduction for mapping and strain construction. For example, let’s say that we have isolated a new mutation in the MotA gene. MotA is a component of the bacterial flagellar moter and MotA– mutants are nonmotile, a phenotype easily detected by the inability of MotA– colonies to “swarm” outward on soft agar plates. Imagine that we want to map the MotA– mutation or to move this mutation into an E. coli strain with a new genetic background. Clearly direct transduction MotA– would not be possible since we have no way to select for rare (1/15,000) transductants with the nonmotile MotA– phenotype. One solution would be to use a nearby marker for which we can select to move MotA– by its cotransduction with the selectable marker. Unfortunately, good selectable markers are not common and we are unlikely to have a good selectable marker placed within cotransduction distance of MotA– readily available. A powerful alternative approach would be to isolate a random Tn5 insertion that is close to MotA– and to use the Kanr trait conferred by Tn5 as the selectable marker for cotransduction