20.109(F09): Sameer Hirji and Adam Rubin - Research Proposal

20.109 Final Project - Research Proposal

 * Engineered bacterial strain lifetime through modification of origin of replication
 * Overview
 * E. coli oriC consists of three AT rich 13-mer repeats and four 9-mer repeats. One could introduce a homologous recombination mechanism to eliminate one repeat on each replication, and thus set the number of replications possible after the introduction of the original genome. This “fail-safe” would prevent the nightmare scenario of runway organisms causing medical or environmental complications.
 * Reference 1
 * Bates D. The right half of the Escherichia coli replication origin is not essential for viability, but facilitates multi-forked replication. Mol Microbiol. 2009 Oct; 74(2):467-79.
 * Summary: Authors found the minimal required sequence for chromosomal OriC. Understanding the limit of OriC manipulation beyond which replication fails is critical to this system.  The OriC can sustain a 163bp deletion and still experience no growth inhibition under normal conditions.
 * Reference 2
 * Katayama T. DnaA structure, function, and dynamics in the initiation at the chromosomal origin. Plasmid. 2009 Sep; 62(2):71-82.
 * Summary: DnaA-ATP binding to OriC is a critical first step in replication complex formation. This binding depends on several key DNA residues, multiple DnaA molecules, and interaction with helicase DnaB.  Any of these mechanisms, especially the critical DNA residues, could be targeted for replication inhibition.
 * Open Questions
 * What mechanisms of natural chromosomal mutation have been observed in bacteria?
 * Is there any memory of replication events stored in the bacterial chromosome?
 * Can high concentrations of intracellular resources be diluted by replication in a predictable fashion?