The occurrence of mutations and their ability to provide an organism with a competitive advantage is central to evolution. Mutations that provide an advantage are relatively rare. The frequency and magnitude of positive, neutral and negative mutational effects is important for the dynamics and outcome of evolutionary processes; however, this distribution of mutational effects has not been comprehensively determined due to experimental limitations. This lack of knowledge has been a major hurdle for the study of how genes and proteins evolve. In this work, engineered bacteria that function analogous to an electrical circuit called a band-pass filter, together with the extensive use of the latest DNA sequencing technology, will be used to determine the distribution of mutational effects for all possible single nucleotide substitutions in a single gene that plays an important role in the evolution of antibiotic resistance. This knowledge will be exploited to address fundamental questions in evolution and practical issues in the application of evolution to biotechnology. Graduate students involved in this research will train undergraduates and high school students through their participation in this project.
This project provided new insights into molecular evolution. One major finding was the first comprehensive study of the effect of mutation on a gene in its native organism. This study addressed competing hypotheses on the origin of the deleterious effects of mutations and the nature of DNA differences at the beginning of genes. In addition, the study provided insight into how the genetic code is arranged and how this arrangement both constrains and facilitates evolution. The study resulted in three papers describing our results, with one more currently in preparation. The research also resulted in a patent application for a new method for DNA mutagenesis that is being licensed by the university to a biotechnology start-up company. The research also provided preliminary evidence for two new research grant proposals currently under consideration at NSF. During the course of this research, three pre-doctoral students, two undergraduates, and one high school student were trained in research. The undergraduates and high school student were trained in molecular biology. The pre-doctoral students received training in molecular biology, synthetic biology, and molecular evolution. Two of the pre-doctoral students presented their research at national meetings. Two pre-doctoral students received experience in training and mentoring the undergraduates and the high school student. The research supported by the grant was incorporated into a class on protein engineering taught by the principle investigator.
