User:Emzodls
20.109 Spring 2007
Last Name
de los Santos
First Name
Emmanuel Lorenzo
Nickname
Emzo
Course/Minor
20
Year of Graduation
2009
emzodls AT mit DOT edu
Subjects this Term
20.109
7.05
6.001
6.041
21F.101
Module 1 : M13 Genome Re-engineering
M13 Redesign Ideas
| Gene | function | design ideas |
|---|---|---|
| I | assembly | modify genes I, IV and XI to allow other useful proteins to attach to the complex (more flexibility in phage modification |
| II | replication of DNA + strand | add some kind of control to deactivate protein (e.g. sensitivity to heat or a chemical stimuli), this allows better control of the phages |
| III | phage tail protein | add myc epitope, or another marker that can be used for experimentation |
| IV | assembly | (see modification for I) |
| V | binds ssDNA | add tag to track changes, change structure to allow more DNA to fit in the phage |
| VI | phage tail protein | bind it tighter to phage body, modify protein to allow more modifications to p3 |
| VII | phage head protein | (see p6), make protein 9 more flexible to modifications |
| VIII | phage coat protein | add myc or another tag (x-ray sensitive, UV sensitive, flourescent) |
| IX | phage head protein | tag, allow p9 to attach to different kinds of proteins including p3 (makes virus chains) |
| X | DNA replication | add another way to control phage propagation |
| XI | assembly | (see modification for I) |
M13.1 Section Design
| Modification/Limitation | Description |
|---|---|
| G10 ORF and G5 promoter | removed overlap between the two sections of code, changed some of the bases in the wobble position (silent mutations) in the ORF randomly to prevent recombination from homologous sequences, added overlapping KpnI and EagI sites. |
| G3 promoter and G8 ORF | similar to g10 ORF, g5 promoter modification, added overlapping SpeI and BclI restriction sites. |
| G7 rbs and G5 ORF | like above, added overlapping ApaI & AvrII restriction sites. |
| (G8 promoter and G9 rbs) and G7 ORF | like above, added overlapping BspEI & BglII restriction sites. |
| (G8 promoter and G9 rbs) and G7 ORF | like above, added overlapping BspEI & BglII restriction sites. |
| G8 RBS and G9 ORF, G9 ORF and G8 ORF | like above, added overlapping NcoI & NarI restriction sites. |
| additional g8 copy | added an aditional g8 copy (promoter, rbs and ORF) this gives two copies of the gene to work with, hopefully each copy is equally expressed in the phage coat, later on, these two copies could be modified to have affinities for two different types of metal allowing us to get an almost equal ratio of materials on the surface of the phage coat. Added overlapping KasI & BsiWI to the ends of this extra gene to make it a relatively reversible transformation. |
| G8 promoter and G9 rbs limitation | i decided not to separate the two as g9 does not have its own promoter and i inferred that the g8 promoter might also function as g9's promoter |
Most of the modifications I made to this section involved removing the overlaps between the gene sections when possible. I did this by first copying the overlapping sequence. I then changed some of the bases at the wobble positions in the open reading frame sections that I duplicated, I made sure that the codon still codes for the same base, this way we can safeguard against recombination. My contribution to the M13 refactoring is the idea of adding another copy of gene 8. Im hoping that both copies are expressed on the cell surface. If this is the case we can modify each copy of the gene independently and make them attract different kinds of materials. This way we can get two different types of material on a single phage.
