This project has succeeded in improving the AlkB gene by random point mutations and by gene shuffling. The directed evolution and screening protocols have been demonstrated to be robust and effective. The screening method using the zones of clearing is very effective at demonstrating degradation and can be scaled up easily in order to screen large numbers of mutants. It has a distinct advantage over some screening methods such as using optical active substrate analogs because when selecting for activity, it selects for improved activity on the desired substrate, some screening methods “addict” enzymes to their specialized substrates.

The limiting factor is that the screening assay is that it is not immediate and requires 48 hours of incubation to determine the activity, also it is often necessary to plate the wild type and the mutants in close proximity in order to observe very minute differences in rates of degradation. The PCR amplification of the AlkB gene was crucial to the project and required a great deal of attention. The yield of the reaction would often be low and many samples had to be pooled in order to have enouph DNA for the digestion and ligations. The optimal conditions were determined by doing multiple gradient reactions, a high annealing temperature greatly reduced the amount of non-specific activity.

Strangely a very large volume of MgCl2 was required, possibly due to the high 65% GC content of Pseudomonas, also at higher Mg concentrations, the non-specific activity changed from a 300bp band to a 1.8kbp band. The gene shuffling is trouble-free and produces large amounts of product. The ligation of the AlkB gene into the pUC 28T vector and the subsequent transformation was problematic and was repeated several times. The rate of mutant A19 is visibly higher then that of the wild type Pseudomonas putida.