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