User:Slokwong/MODULE 1 : Genome Engineering

Notes on M13 Renovation

M13 genome

1. Overlapping regions:
   • g2 and g10 (496-831)
   • g9 and g8 (1301-1304)
   • g7 and g9 (1206-1209)
   • g1 and g11 (3916-4242) – g11 is contained in g1, share STOP codon
   • g1, g4, and g11 (4220-4242)
2. Independent genes:
   • g3, g5, g6

Restriction enzymes used

• Pst1 digest

M13.1 Design

Gene	Function	Re-engineering Idea
I	assembly	Modify so that p1 can interact with multiple p4. Then analyze how phage secretion changes. Changing p1-p4 interaction affects how effective the channels are.
II	replication	Modify such that it nicks the DNA in a way that helps p5 sequester the + strands more effectively, thus controlling the formation of double stranded DNA and perhaps making p5 work more efficiently by reducing competition. Be careful in modifying because it is linked to g10.
III	phage tail protein (5 copies)	Add myc or alternative tag to monitor the time progression of the phage escape from host.
IV	assembly	Modify in a way that increases p4 affinity for p1. This makes the channels more effective.
V	binds ssDNA	Modify such that p5 can sequester the + stranded DNA more effectively so that there is less competition with the formation of double stranded DNA.
VI	phage tail protein (5 copies)	Since p6 is the accessory protein to p3, it can be modified so that p3 can interact more effectively with the host.
VII	phage head protein (5 copies)	Since p7 is the companion protein of p9, make same modifications as that of p9.
VIII	phage coat protein (2700 copies)	Add myc or alternative tag to aid in targeting various types of hosts.
IX	phage head protein (5 copies)	Modify in such a way to make the phage secretion occur at a faster rate so that interaction time with the host is reduced. Also, modify so that p9 interaction with p5 is more effective. Since part of g9 overlaps with g8, be careful in making modifications.
X	replication	Modify such that the + strands of DNA are not soley dependent on the presence of p10. This modification works together with our modification of p2.
XI	assembly	Same modification as g1.

I chose to redesign the region that contains g3. In order to destroy the overlapping regions of the promoter of g3 and the ORF of g8, I mutated two base pairs in wobble positions of the g8 ORF so that the g3 promoter cannot exist in the same region. I did this without changing the amino acid sequence. Also, I chose to modify the g8 ORF instead of the g3 promoter because I was unsure of what functions in the promoter I would be disrupting. Then I duplicated the g3 promoter and added it to the end of g8 with two restriction enzymes ecoRI and XmaI. These restriction enzymes create sticky ends that help recognize the orientation of the DNA sequence. As a Designer, I wanted to find out what new function is created when a g3 ORF is imbedded in another g3 ORF. Thus, I cut the region between g3 promoter and g3 RBS with the restriction enzyme NlaIV. Because this enzyme creates a blunt end, I can’t determine whether the DNA was inserted in the correct orientation. However, I will know it will not work if there is no expression of g3 because the promoter g3 is upstream of the inserted region while the existing g3 ORF is downstream of it. Having duplicated g3 inside of another g3 ORF, I want to see whether there are new functions created, such as the effects on the timing of the phage escape from the host.

Preview M13.1 here! [[1]]

Modification	Description
g8 ORF & g3 promoter	Destroyed overlapping regions by mutating two base pairs in g8 ORF, thus g3 promoter cannot exist in same region. Then add ecoRI and XmaI restriction enzymes so that it will be easier to make future modifications. These also serve to help orient the DNA.
g3 promoter & ORF	Duplicated them as a result of unstuffing.
g3 ORF & RBS	Duplicated them and then imbedded them into the region between the g3 promoter and RBS sites. As a result, a g3 is imbedded into another g3.