User:Msoh

M13 Reengineering

Gene	Ideas
I	influence how it assembles and interacts with IV to alter size/properties of channels, this could be important if the size or shape of the phage is changed
II	make it sensitive to a mechanism that can control the proliferation of the phage, perhaps make it require a cofactor that must be added before replication begins
III	present larger molecules (including myc epitope), change its interactions with bacterial surface molecules to influence which phages are able to replicate, change which bacteria the phage is able to interact with
IV	influence its assembly and interaction with I/XI to alter size/properties of channels
V	make it sensitive to mechanism that allows for assay of DNA amount and location, change its assembly mechanism to influence phage size
VI	change the way it interacts to influence III's binding affinities
VII	change the way it interacts to influence IX's binding affinities
VIII	present small molecules (such as myc epitope), regulate size of phage or influence shape of phage by changing how it assembles into a coat, this could involve changing its interactions with V
IX	present larger molecules, influence the way DNA is packaged, perhaps thereby controlling proliferation
X	changes to II will cause changes to X, perhaps a dual control mechanism
XI	changes to I's interactions with IV will automatically change XI

M13 Refactoring

I approached the refactoring of the M13 phage with a design perspective. My aim was to render the M13.1 refactored phage as easy to manipulate as possible for future engineering designs. This involved eliminating the many overlaps between open reading frames and control sequences. Using the model provided by “Refactoring T7” (Chan, et al., 2005), I duplicated overlapping sequences and separated them by unique restriction sites. The duplicate sequences in the ORFs were modified by silent codon mutations. As opposed to the Chan methodology, I only included one restriction site between each sequence for efficiency purposes. I defined each part, to be divided by restriction sites, as either a promoter or an RBS/ORF complex. My focus in designing was on the promoters, as I am most interested in manipulation of protein expression levels in the phage. I began this engineering goal by including a ptacI promoter to regulate g8, which allows for direct expression level control of g8 and eliminates the need for the highly overlapping g8 promoter. I chose g8 because I felt it was the best candidate for phage display engineering projects based on Prof. Belcher’s nanomaterials research and in consideration of M13’s small size.

Part Name	Change	Reasoning
M31372 (g2)	-region before HpaI eliminated -gcataa to gcTtaa at end of reading frame -starting bp 129, sequence reads aaAtgggaGtcaacAgtt	-limit scope of refactoring -eliminate direct repeats due to RBS g5 replication -eliminate direct repeats due to Promoter g5 replication
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
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
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

Registry Number: BBa_M31270