Module 1, Day 1

M13 Re-Engineering Ideas

Protein	Function	Re-engineering Ideas
I	Assembly: bacterial inner membrane (secretion channels)	P1, P4, and P11 are inextricably linked in both genetic sequence as well as function. It would be interesting to see how mutating sequences within this genomic region affects the characteristics of the secretion channels--can we change the size of the channels to allow different sized phages to pass through? If the channels are too small, will the bacterial cell lyse as a result of the trapped viral particles, or will the virus particles just die off? Can we manipulate the speed and/or efficacy of viral release through the channels? Also, will altering a non-overlapping segment of one of these proteins result destroy the teamwork of the three proteins, or can the other proteins adapt? Similarly then, will altering an overlapping sequence (ie: around 4230) result in proteins that are altered in a way that still allows them to function together (because they will have been changed in the same way)? Playing around with these proteins can offer insight into the organization and function of overlapping genes. It could also be interesting to delete proteins 1 or 11 and see if the secretion channels would still form--are they functionally redundant?
II	Replication of DNA + strand	Would not want to mess with this protein too much and disrupt the genomic replication process.
III	Phage tail protein (5 copies)- rounded end: first point of infection, last point of contact. interacts with TolA on bacterial pilus	Perhaps this protein could be re-engineered to recognize certain antibodies that would allow M13 to be more easily "marked" or tracked. This could also be used to target viral infection towards certain types of cells. Also, it has been found that deletion of P3 results in inability of M13 to leave its host. Can we alter P3 so that it is unable to infect the host in the first place? Or would such a genome never propagate in the first place?
IV	Assembly: forms barrel like structures in outer bacterial membrane (secretion channels)	See PI
V	Binds ssDNA: produces protein/DNA complexes designed for packaging into new phage particles	Would also not mess too much with this protein. Perhaps add a cleavage site before the start codon, as there are not too many cleavage sites around this section of the genome. If we subdue the protein's function of sequestering of single stranded genomes, will the virus be packaged with double stranded DNA?
VI	Phage tail protein (5 copies)- accessory to P3	Currently an accessory tail protein, possibly because of low synthesis levels due to "G" following the ATG initiation codon. Mutate this codon from "G" to "A" and observe if translation activity is increased--will this result in more than 5 copies of the tail? Perhaps also mutate to facilitate or block M13's passage through secretion
VII	Phage head protein (5 copies)- more buried	Currently an accessory head protein, possibly because of low synthesis levels due to "G" following the ATG initiation codon. Mutate this codon from "G" to "A" and observe if translation activity is increased--will this result in more than 5 copies of the head?
VIII	Phage coat protein (2700 copies)- flexible enough to adjust to the size of the genome	Perhaps delete this gene to see if P5 is capable of maintaining the capsid on its own, or alter its composition to see if other, potentially useful materials can be "grown" as viral coding.
IX	Phage head protein (5 copies)- "blunt end"	If we alter this protein to the same rounded structure as in the tail, could the virus enter and exit bacterial cells from either end? Can we alter the shape / properties of the head so that it does not require secretion channels to exit the cell, and can utilize natural channels in the bacterial membrane?
X	DNA replication: regulates # of double stranded viral genomes in the host; linked to p2	Can we alter this protein without altering P2? If so, we could toggle the protein to give us control of accumulation of viral genomes within the bacterial cell.
XI	Assembly: bacterial inner membrane (secretion channels)	See PI

M13's Closest Relatives

M13 is a small, filamentous bacteriophage classified under Group II of the DNA viruses. Viruses in this group are characterized by having genomic information stored in a single strand of DNA (ssDNA), and includes the Parvoviridae family. M13 differs from most others in this family in its temperate infection of a host cell that does not result in the immediate lysis and destruction of the host. Amongst its closest evolutionary relatives in this group are fd and f1, differing from each by only a few nucleotides in length, with approx. 3% difference in base pairs.

"BBa_M1307 is not a standard biological part and does not belong in the registry"

Though Bba_M1307 refers to an entire genome and not a traditionally smaller genetic "brick," it can also be argued to be a standard biological part because it has a set function of providing phage coat proteins for other M13 viruses that are taking in plasmid inserts. Thus, while not physically as sound as most parts in the registry, its functional composition seems to pass the test.