Katpak:m1d1

Module 1 Day 1: Start-up genome engineering

1. Generate a table that lists each gene and any re-engineering ideas you have for it.

M13 Genes

Gene	Location	Function	Notes	Re-Engineering Ideas
I	3196-4242	assembly, forms channel in inner membrane	overlaps with gene IV and geneXI, has the same stop codon as geneXI	add a regulating element in front of promoter to test effects of increaseing or decreasing number of channels, add an element that can regulate when the channel can open or close based on concentration, temperature, pH, concentration of phage, or other environmental factors
II	8268-831	knicks dsDNA for replication of + strand	geneX overlaps with C-terminal of geneII	add a detection mechanism to nick the + strands of only certain phages--with a specific inserted recognizable gene sequence, for instance
III	1579-2853	phage tail protein (5 copies), contacts TolA on pilus	Contains BamHI site	modify to bind to a protein other than TolA on the bacterial pilus, create a way to control binding strength
IV	4220-5500	assembly, forms channel in inner membrane	overlaps with geneI and geneXI	add a regulating element in front of promoter to test effects of increaseing or decreasing number of channels, add an element that can regulate when the channel can open or close based on concentration, temperature, pH, concentration of phage, or other environmental factors
V	843-1106	binds ssDNA to make protein/DNA complex for packaging		add elements to increase affinity to + stranded DNA, alter packaging sequence to increase interaction to protein VIII or to change interaction to another type of protein or perhaps an inorganic material
VI	2856-3194	phage tail protein (5 copies), forms cap with proteinIII		modify to bind to a protein other than TolA on the bacterial pilus, create a way to control binding strength, add a GFP fusion
VII	1108-1209	blunt end of phage,interacts with protein V	stop codon overlaps with geneIX start codon	modify to allow for integratable insertion of various elements, make fusion to GFP
VIII	1301-1522	phage coat protein (2700 copies)	start codon overlaps with geneIX stop codon	modify to allow for integratable insertion of various elements, make fusion to GFP, add other surface proteins that affect solubility in the bacterial membrane, add ability to asseble or interact with inorganic materials
IX	1206-1304	blunt end of phage,interacts with protein V (5 copies)	start codon overlaps with geneVII stop codon,stop codon overlaps with geneVIII start codon	modify to allow for integratable insertion of various elements, make fusion to GFP, add an element to see if motility can be increased such as a flagella-like motor
X	496-831	allows + strand accumulation/regulation	overlaps with C-terminal of geneII	modify to increase accumulation of double stranded genomes in bacterial host
XI	3916-4242	assembly, forms channel in inner membrane	overlaps with geneI and geneIV, has the same stop codon as geneI	add a regulating element in front of promoter to test effects of increaseing or decreasing number of channels, add an element that can regulate when the channel can open or close based on concentration, temperature, pH, concentration of phage, or other environmental factors

2. Nature often preserves functionally critical genomic elements, and evolutionary cousins can help us identify which genetic elements are disposable, which are interchangeable, and which are essential. Who are M13's closest evolutionary relatives and how do they differ from the phage you're working with?
M13's closest evolutionary cousins are the other small, filamentous, male-specific coliphages--fd and f1. These phages are characterized by a small, single-stranded circular DNA genome of about 6400 bases. There is an average of 3.0% of nucleotide-sequence differences between the M13 and fd genomes. Only 12 of the changes result in change of amino acids. M13 DNA is only a single nucleotide shorter than fd DNA.