Part:BBa_M31270

Refactored M13K07 Phage Genome

I approached the refactoring of the M13 phage with a design perspective. My aim was to render the M13.1 refactored phage as easy to manipulate as possible for future engineering designs. This involved eliminating the many overlaps between open reading frames and control sequences. Using the model provided by “Refactoring T7” (Chan, et al., 2005), I duplicated overlapping sequences and separated them by unique restriction sites. The duplicate sequences in the ORFs were modified by silent codon mutations. As opposed to the Chan methodology, I only included one restriction site between each sequence for efficiency purposes. I defined each part, to be divided by restriction sites, as either a promoter or an RBS/ORF complex. My focus in designing was on the promoters, as I am most interested in manipulation of protein expression levels in the phage. I began this engineering goal by including a ptacI promoter to regulate g8, which allows for direct expression level control of g8 and eliminates the need for the highly overlapping g8 promoter. I chose g8 because I felt it was the best candidate for phage display engineering projects based on Prof. Belcher’s nanomaterials research and in consideration of M13’s small size.