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. The team was advised by three graduate students and three faculty mentors.
The goal of our project is to create a λ bacteriophage that can selectively lyse specific subpopulations of E. coli. We will first use recombineering techniques to insert amber mutations into three key developmental genes in λ-Zap. Next, a second copy of these genes, controlled by a cis-repressing riboregulator, will be cloned into the phage genome at the ribosome binding site upstream of each of the three critical genes, thus blocking the expression of key viral developmental proteins. As depicted in the diagram below, the expression of trans-activating RNA in the target bacterial host will relieve the repression by opening up the ribosome binding site, enable the translation of the viral developmental gene and allow lysis of the host cell. Hosts which do not contain this RNA will remain intact.
Currently, E. coli strains have been constructed that contain a low-copy plasmid construct where one of three key developmental viral genes - coding for the Cro, N, or Q proteins - 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. Heterologous N and Q have been shown to complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q and prevented complementation. We were unable to express sufficient cro from a plasmid to rescue a cro mutant phage.
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 N and Q amber mutants.