IGEM:Caltech/2007/Project Description

The Caltech iGEM 2007 team is composed of four Caltech undergraduates and one undergraduate from MIT. Team members are current juniors and seniors in biology, chemistry, chemical engineering, and biological engineering. In addition, three graduate student advisors, as well as three faculty advisors are involved in helping the progress of the team's work. The team began meeting in April for preliminary discussions and brainstorming on possible projects. In May, we voted on the project idea of most interest to each member. The decision to work with lambda phage infection was unanimous. Each team member felt that this was a fascinating project from the standpoint of synthetic biology because it challenged us to control the complex process of viral infection. In addition, we felt that the project held relevant medical applications in promising to add a knowledge-base to the field of gene therapy.

The goal of our project is to create a λ bacteriophage that can selectively infect E. coli by knowing which cells to lyse. We aim to do this by first using recombineering techniques to knock out three key developmental genes in λ-Zap phage, which is a great cloning vector; amber point mutations will thus be introduced in wild type loci of these genes. Thereafter, a second copy of these genes will be cloned in with a cis-repressing riboregulator elsewhere in the viral genome. The expression of trans-activating RNA, moreover, will not only relieve repression but will also rescue the crippled phage. Currently, Escherichia coli strains have been cloned to contain a plasmid construct where one of the three key developmental viral genes, which encode for Cro, N, or Q protein, is regulated by a tetracycline-dependent promoter. The addition of anhydrotetracycline (aTc) inactivates the tetracycline repressor and leads to the production of the respective viral protein in the E. coli cells. This allows us to control the concentration of viral protein produced in the cells by adding varying amounts of aTc to the bacterial growth media. Lysis curves are being created for varying aTc amounts, so that the effects of protein concentration on the switch between lysis and lysogeny can be better understood. Multiple riboregulator designs are being tested (for both activation and repression levels), and successful designs will be cloned into the plasmid constructs. Phages resulting from the recombineering process are also being screened for successful cro/N/Q amber mutants.