Investigations into Open Notebook Science: directed evolution as a model for exploring online laboratory notebooks.

Abstract

Directed evolution is used by protein engineers to alter the properties of an enzyme and can be considered as an uphill walk through a fitness landscape. The process is used to mimic natural evolution, though natural adaptive evolution is poorly understood. There are currently several linked theories as to how adaptive evolution occurs: through adaptation of the promiscuous functions of enzymes, conformational dynamism and neutral drift. Neutral drift is the incorporation of mutations that have little or no effect on the protein in its current environment but which may have adaptive potential if the selection pressure changes. Neutral drift may also provide alternative routes through the fitness landscape of a protein that allows it to reach new peaks of fitness not accessible by direct selection. In this thesis the question of whether neutral drift is a useful technique to employ in directed evolution is explored. The E. coli ?-glucuronidase is evolved into a ?-galactosidase in four rounds of neutral drift, i.e. selecting for the native activity. Across four experiments (not all carried out as part of work for this thesis), five key mutations are returned: T509A, S557P, N566S, K568Q and W529L. These mutations are identical to mutations found in standard directed evolution experiments on this enzyme system. The libraries of mutant ?-glucuronidases were screened against a variety of alternative substrates and three mutants with ?-xylosidase activity were found. These are different mutations to those found in other experiments. This thesis also explores electronic methods of recording research data including ELNs, blogs and wikis. The chapter also features the development and testing of a blog system named LaBlog. The experiments using neutral drift are used to develop and optimise the blog for molecular biology type work; though the system is flexible and can be optimised for other types of scientific work. Additionally blogs are compared to wikis, another online recording method, in the context of the Open Notebook Science Challenge: a challenge to measure the solubilities of compounds in organic solvents.

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