User:Nzimm: Difference between revisions
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====M13K07 Genome Refactoring:==== | ====M13K07 Genome Refactoring:==== | ||
In refactoring the M13K07 genome between the HpaI site in gene II and the BamHI site in gene III, I separated all overlapping elements across the region by copying overlapping sequences and placing the elements adjacent to one another on the refactored sequence. As a result, the refactored region is 1,000 bp larger than the original sequence. I also added annotation for & refactored the open reading frames of genes 2, 5, 7, and 10, which are not annotated in the M1307 part in the registry. I intend to separate each element with two unique restriction sites, so that the elements can be isolated & manipulated more easily. Below is a table describing the overlaps that were refactored. | In refactoring the M13K07 genome between the HpaI site in gene II and the BamHI site in gene III, I separated all overlapping elements across the region by copying overlapping sequences and placing the elements adjacent to one another on the refactored sequence. As a result, the refactored region is 1,000 bp larger than the original sequence. I also added annotation for & refactored the open reading frames of genes 2, 5, 7, and 10, which are not annotated in the M1307 part in the registry. I inserted the sequence encoding the myc protein into g3. I intend to separate each element with two unique restriction sites, so that the elements can be isolated & manipulated more easily. I initially proposed to add a fluorescent tag like GFP to g5, but seeing as how GFP is nearly 3 times the size of the protein product of g5, I chose to leave g5 unaltered. Below is a table describing the overlaps that were refactored. | ||
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Revision as of 01:19, 27 February 2007
Neil Zimmerman
Bio
- MIT Class of 2009
- Major: Biological Engineering
- Minors: Management and Music
- Varsity Soccer
- Wind Ensemble (Clarinet)
20.109
M13K07 Genome Engineering Plans:
| Gene | Plans |
|---|---|
| I | Change # of protein copies expressed to experiment with different sized channels |
| II | Modify residues to allow deactivation of p2 under certain conditions so that replication of + strand can be regulated |
| III | Insert myc to allow detection with an antibody |
| IV | Change # of protein copies expressed to experiment with different sized channels |
| V | Add fluorescent tag to monitor levels of p5-ssDNA complex |
| VI | Modify residues to help p3 bind more effectively to the ToIA protein on the E. coli F pilus |
| VII | Minimize the bulk of the protein to allow more room for modifications on p9 |
| VIII | Insert myc (as in III) or another tag to serve as a “hook” for attaching constructs to M13 |
| IX | Add residues to N-terminus to present on the outside of the phage coat |
| X | Add sensitivity to different stimulus than that of p2 in order to regulate replication of + strand in another fashion |
| XI | Modify residues to allow proteins other than p8 to embed in the membrane and serve as the phage filament coat |
M13K07 Genome Refactoring:
In refactoring the M13K07 genome between the HpaI site in gene II and the BamHI site in gene III, I separated all overlapping elements across the region by copying overlapping sequences and placing the elements adjacent to one another on the refactored sequence. As a result, the refactored region is 1,000 bp larger than the original sequence. I also added annotation for & refactored the open reading frames of genes 2, 5, 7, and 10, which are not annotated in the M1307 part in the registry. I inserted the sequence encoding the myc protein into g3. I intend to separate each element with two unique restriction sites, so that the elements can be isolated & manipulated more easily. I initially proposed to add a fluorescent tag like GFP to g5, but seeing as how GFP is nearly 3 times the size of the protein product of g5, I chose to leave g5 unaltered. Below is a table describing the overlaps that were refactored.
| Overlapping elements | Action |
|---|---|
| rbs g7 & g5 ORF | copied overlapping sequence & placed rbs g7 adjacent to the 3' end of the g5 ORF |
| promoter g8 & rbs g9 | copied overlapping sequence & placed rbs g9 adjacent to the 3' end of promoter g8 |
| promoter g3 & g8 ORF | copied overlapping sqeuence & placed promoter g3 adjacent to the 3' end of g8 ORF |
| g10 ORF, g10 rbs, g10 promoter, g5 promoter, & g2 ORF | copied g2 ORF & placed adjacent to 5' end of other (non-overlapping) elements |
| inserted the sequence encoding the myc protein into the BamHI site of g3 |
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Email: nzimm (at) mit (dot) edu
