User:Alji: Difference between revisions
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==== M13 Refactoring ==== | ==== M13 Refactoring ==== | ||
I attempted to refactor the M13K07 phage by removing the overlapping areas of genes between the HpaI site of gene 2 and the BamHI site of gene 3. I did this by duplicating each gene (promoter, rbs, and ORF) and placing it after the ORF of the previous gene. I mutated the RBS's of each overlapping area to a silent mutation to prevent readout of the overlapping sections. There were 6 places of overlap, and I have included what changes I made. As a result, I have added 769 bp to the original section of DNA. In between each gene, I left 11 extra base pairs in order to introduce restriction sites so that each part can be manipulated individually. I want to see what others have done before attempting to introduce new restriction endonuclease sites. | |||
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Revision as of 10:53, 28 February 2007
Andrew Ji
About Me
- Year: 2009
- Major: Biological Engineering
- Minor: Economics
- Email: alji AT mit DOT edu
M13K07 Genome Re-engineering ideas:
Gene | Ideas |
---|---|
I | Change size of protein to see effect of different channel sizes. |
II | Increase or decrease expression to optimize rate of + strand replication. |
III | Myc tag to monitor expression in phage and bacteria or to add other things to test initial interaction with host. |
IV | Change size of protein to see effect of different channel sizes. |
V | Add more DNA binding sites to see if more DNA can be packaged into phage. |
VI | Myc tag to monitor expression in phage and bacteria or to add other things to test initial interaction with host. |
VII | Tag protein to monitor interaction with p5/DNA complex. |
VIII | Change size of protein to experiment with the size of coat. |
IX | Tag protein to monitor interaction with p5/DNA complex. |
X | Increase expression to see if more phage leave the host E. coli. |
XI | Change size of protein to see effect of different channel sizes. |
M13 Refactoring
I attempted to refactor the M13K07 phage by removing the overlapping areas of genes between the HpaI site of gene 2 and the BamHI site of gene 3. I did this by duplicating each gene (promoter, rbs, and ORF) and placing it after the ORF of the previous gene. I mutated the RBS's of each overlapping area to a silent mutation to prevent readout of the overlapping sections. There were 6 places of overlap, and I have included what changes I made. As a result, I have added 769 bp to the original section of DNA. In between each gene, I left 11 extra base pairs in order to introduce restriction sites so that each part can be manipulated individually. I want to see what others have done before attempting to introduce new restriction endonuclease sites.
Overlapping Areas | Original Sequence | Modified sequence |
---|---|---|
Gene 2 ORF and Gene 10 RBS | At bp 478: gcatttgag | gcGtttgag |
Gene 2 ORF and Gene 5 RBS | At bp 826: gcataaggt | gcGtaaggt |
Gene 10 ORF and Gene 5 RBS | At bp 1286: gcataaggt | gcGtaaggt |
Gene 5 ORF and Gene 7 RBS | At bp: 1609: gttccggct | gtCccggct |
Gene 7 ORF and Gene 9 RBS | At bp: 1733: cgctggggg | cgAtggggg |
Gene 9 ORF and Gene 8 RBS | At bp: 1869: aatggaaac | aaCggaaac |
The refactored genome is Part BBa_M31332 on the Registry of Standard Biological Parts