If you wanted to discover a novel biosynthetic pathway for an antimicrobial compound and were given a handful of soil, describe the key experimental steps you would need to take to accomplish the task and explain why alternative procedures were not perfor

Question

What will be an ideal response?

Answer 1

Answer: Environmental gene mining steps are illustrated in Figure 11.33 of the textbook. DNA rather than RNA would be directly isolated to ensure capturing any not expressed at that particular instance. Larger expression vectors would need to be selected to house a potential series biosynthetic genes altogether. DNA cut to approximately 40 kbp in a fosmid would be suitable for this study, and the inserts would be ligated into the vectors. The library would be transfected into a compatible host so that the population of cells represents numerous discrete spans of DNA that can then be screened for the function of interest-antimicrobial. If the antimicrobial is of broad spectrum it might even kill its own expression host after induction but confirmation would need to be screened by taking the spend medium and spotting it onto other microorganisms to visual growth inhibition (i.e., zones of clearing) on semisolid nutrient agar plates. For those transfected and produced an inhibitory compound in the medium, sequencing of the vector would not elucidate the genes responsible for it necessarily due to the insert size. Mutagenesis of the cosmid to observe the loss of function and sequencing to identify the displaced gene would enable identification of the genes responsible for the antimicrobial biosynthesis.