Future potential of directed evolution to hydrocarbon degradation as expounded in this project implies an increased number of mutants screened for improved activity, and development of a multi-enzyme complex. Engineering of the entire pathway is necessary at some point in the engineering of the AlkB gene, due to a barrier to improving remediation with the manipulation of a single enzyme. Otherwise, further improvement of AlkB towards large alkanes eventually leads to its products not recognizable by other enzymes in the pathway.

With future methods it is hoped that entire pathways could be mutated in one pass - especially an operon organized pathway such as Alk. Furthermore, the bioremediation application raises questions of toxicity of substrates and diffusion limitations to bacteriology.

Applying genetically engineered microorganisms into the environment is an ethical and public challenge to any forthcoming industrial application. We proposed that application of engineered enzymes is preferential among microbiological products not only for methodology but also in that it is non-living substance controllable with fewer uncertainties and a spontaneous degradation effect. Directed evolution looks to have an important role in the future of any field wishing to optimize biochemical transformation of compounds. There is great potential in hydrocarbon remediation at small energy cost.

The potential of Pseudomonas putida in degrading high-molecular-weight alkanes can be improved by a combination of errorprone Polymerase Chain Reaction amplification and DNase I shuffling of the Alkane Monooxygenase gene (AlkB). Methodical plate screening showed a pronounced effect with a significant range of substrates, with improvement in the rate of degradation by transformation with pUC 28T on AlkB construct likely due to increased AlkB expression with high copy number of the construct plasmid. Among factors limiting scalability were delay of incubation and necessity to plate wild types and mutants in close proximity for rate differentiation. Low reaction yields led to samples pooled to collect DNA for digestion and ligations and a larger volume of MgCl2 than expected was required.

In conclusion, the objective of improving the AlkB gene was met. Robustness of the zones of clearing screening method effectively demonstrated scalable degradation with no enzymatic addiction to substrates. Amplification by PCR and gene shuffling of the AlkB gene was successful. Optimal conditions were determined by multiple gradient reactions; a high annealing temperature reduced the extent of non-specific activity. At higher Mg concentrations, the change from a 300bp band to a 1.8kbp band in non-specific activity was notable. The rate of degradation of mutant A19 was satisfactorily higher than that of the wild type Pseudomonas putida.