The development and application of a random mutagenic technique for the mutation and directed evolution of Reverse Transcriptase

Directed evolution is a powerful tool in the arsenal of the molecular biologist. It provides scientists with the ability to harness the power of evolution. The significance of the discovery of directed evolution was recognized by the award of the 2018 Nobel prize in 2018 to Frances Arnold.
Reverse transcriptase is an enzyme with many different functions, foremost among them being RNA dependent DNA polymerisation. This function has made reverse transcriptase a key enzyme for both molecular biology research and diagnostic applications. There is a demand for reverse transcriptase with increased functionality, be it increased thermotolerance enabling the enzyme to retain activity at temperatures above the denaturation point of RNA, or increased terminal transferase activity, to better facilitate template switching in vitro. Reverse transcriptase is therefore an ideal candidate for directed evolution.
In the course of this work, a novel statistical model for the introduction of mutations over the course of an error prone polymerase chain reaction is described. This model is then validated by the mutagenesis and subsequent sequencing of three model systems: an engineered form of a bacteria rubredoxin protein, reverse transcriptase, and a polyethylene terephthalate dehydrogenase. The gene encoding reverse transcriptase is mutagenised and expressed in a recombinant form, the resultant mutants are assayed for increased RNA-dependent DNA-polymerase activity at 65℃. The work carried out in this thesis represents an attempt to better understand the process by which mutations are introduced via error-prone DNA polymerization. It is posited how these advancements can help to minimise the number of rounds of directed evolution that are required before an appropriate end point is reached.