Melee:20.109 stuff
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20.109 Stuff
Module 4 Research Proposal
SAGA Subunits, S. cerevisiae
M13 phage re-engineering ideas:
| Gene | Ideas |
|---|---|
| I | Modify complex such that proteins other than p8 may assemble onto the coat. |
| II | Make p2 sensitive to various stimuli (e.g. heat, light) so replication can be regulated. |
| III | Add myc, or other tags. Change residue sequence to create affinity for different materials. |
| IV | Modify complex such that proteins other than p8 may assemble onto the coat. |
| V | Add a tag different from p8 (e.g. RFP) to determine what stage of the phage life cycle it is in. |
| VI | Modify p8 such that it binds to p3 in a way to allow more flexibility in modifying p3. |
| VII | Like, p6, modify to make p9 more flexible. |
| VIII | Add myc, or other tags, e.g. GFP. |
| IX | Modify p9 to bind to p3 to create long filaments of phage lined up end to end. |
| X | Make p10 sensitive to a different stimulus than p2 to again regulate replication. |
| XI | Modify complex such that proteins other than p8 may assemble onto the coat. |
M13 Refactoring
I attempted to remove all overlapping sites in this section of the genome. What I did was copy the overlapping section (usually an RBS and/or promoter overlapping the previous ORF) and repeat it. I changed the wobble positions of every other codon on the ORF’s so that they wouldn’t homologously recombine with the repeated sequence. Additionally, as was done on the T7.1 refactoring, I added two new cut sites between the separated genes. I chose to keep the overlap between the gene VIII promoter and gene IX RBS (and the resulting mixed order of genetic elements) changing that order seemed to be a much more significant change than the others.
| Overlapping Areas | Added Restriction Sites | Modified sequence |
|---|---|---|
| Gene V Promoter/RBS - Gene X ORF | AflII (CTTAAG); ApaI (GGGCCC) | ccaacgAcctgaTtggtaCaatgaAccagtActtaaGatcgcCtaaCTTAA GGGCCCaacgtcctgactggtataatgagccagttcttaaaatcgcataa |
| Gene VII RBS - Gene V ORF | BmtI (GCTAGC); BssHII (GCGCGC) | gttccAgctaaAtaaGCTAGCGCGCgttccggctaagtaa |
| Gene VIII Promoter/IX RBS/ORF – Gene VII ORF | KpnI (GGTACC); NcoI (CCATGG) | aatTtccgtCgtactCtgtttTgcgctCggtatTatcgcCgggggCcaaagGtga GGTACCATGGaatctccgttgtactttgtttcgcgcttggtataatcgctgggggtcaaag |
| Gene VIII RBS/ORF – Gene IX ORF | NheI (GCTAGC); MfeI (CAATTG) | taatGgaaacCtcctcTtgaGCTAGCAATTGtaatggaaacttcctcatga |
| Gene III Promoter – Gene VIII ORF | BclI (TGATCA); BglII (AGATCT) | aattcacTtcgaaGgcaagTtgaTGATCAGATCTaattcacctcgaaagcaagctga |
The refactored genome is Part:BBa_M31511 on the Registry of Standard Biological Parts
