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 duplication -eliminate direct repeats due to Promoter g5 duplication
M31380	ApaI restriction site inserted (GGGCCC)	partition sections for manipulation
M13105 (Promoter g5)	moved out of g2 ORF	no more overlapping sequence
M13505 (RBS g5)	moved out of g2 ORF	no more overlapping sequence
M31375 (g5)	end of reading frame now reads gtAccggcAaaCtaa	eliminate direct repeats due to RBS g7 duplication
M31381	BssHII restriction site inserted (GCGCGC)	partition sections for manipulation
M13507 (RBS g7)	moved out of g5 ORF	no more overlapping sequence
M31377 (g7)	starting bp 49, sequences reads atTtccgtAgtactAtgtttGgcgctAggtatTatcgcAgggggAcaaagGtga	eliminate direct repeats due to RBS g9 duplication; also removes the g8 promoter sequence
M31382	EcoRI restriction site inserted (GAATTC)	partition sections for manipulation
M13509 (RBS g9)	moved out of g7 ORF	no more overlapping sequence
M31379 (g9)	end of reading frame now reads atggaGacttcGtcTtga	eliminate direct repeats due to RBS g8 duplication
M31383	NcoI restriction site inserted (CCATGG)	partition sections for manipulation
M31370 (tacI)	inserted tacI promoter sequence (Boer et al., 1983)	phage titers done in E. coli K12 ER2267 strain, which is laqIq so is compatible with promoter; promoter is directly regulated by concentration of IPTG
M13509 (RBS g8)	moved out of g9 ORF	no more overlapping sequence
M31378 (g8)	end of reading frame now reads ttTacctcCaaagcTagTtga	eliminate direct repeats due to Promoter g3 duplication
M31384	XbaI restriction site inserted (TCTAGA) bounded by naturally occurring thymines	partition sections for manipulation; maintain promoter-RBS distance
M13103 (Promoter g3))	moved out of g8 ORF	no more overlapping sequence
M13503 (RBS g3)	-	-
M31373 (g3)	stopped at BamHI site	limit scope of refactoring

Registry Number: BBa_M31270

SAGA subunits, S. cerevisiae

components of the Ada histone acetyltransferase complex

Ada subunits	size,chromosome,null p-type	notes
Ada1 (aka HFI1, SUP110, SRM12, GAN1)	1.467 kb/489 aa, Chr. XVI, viable	used for structural integrity, interacts with H2A, role in cell structure and respiratory processes
Ada2 (aka SWI8)	1.305 kb/434aa, Chr. IV, viable	transcription coactivator, role in sporulation and drug resistance
Ada3(aka NGG1, SWI7)	2.109 kb/702aa, Chr. IV, viable	glucose repression of Gal4p-regulated genes, role in drug resistance
Gcn5 (aka ADA4, SWI9)	1.32 kb/439aa, Chr. VII, viable	catalytic role as histone acetyltransferase, acetylates N-terminal lysines in H3 and H2B, role in DNA repair
Ada5 (aka SPT20)	1.815 kb/604aa, Chr. XV, viable	structural integrity of SAGA complex, role in drug resistance

suppresses Ty insertion mutations

Spt subunits	size, chromosome, null p-type	notes
Spt3	1.014 kb/337aa, Chr. IV, viable	activates RNA Polymerase II-dependent genes, controls other promoters
Spt7(aka GIT2)	3.999 kb/1332aa, Chr. II, viable	helps assemble the SAGA complex
Spt8	1.809 kb/602aa, Chr. XII, viable	helps SAGA inhibit some promoters, role in trp and chitin metabolism
Spt20 (aka Ada5)	1.815 kb/604aa, Chr. XV, viable	subunit involved in structural integrity

TATA binding protein-Associated Factors

TAF subunits	size, chromosome, null p-type	notes
TAF5 (aka TAF90)	2.397 kb/798aa, Chr. II, inviable	involved in RNA polymerase II transcription initiation and in chromatin modification
TAF6 (aka TAF60)	1.551 kb/516aa, Chr. VII, inviable	involved in RNA polymerase II transcription initiation and in chromatin modification, H4 analogue
TAF9 (aka TAF17)	0.474 kb/157aa, Chr. XIII, inviable	involved in RNA polymerase II transcription initiation and in chromatin modification, H3 analogue
TAF10 (aka TAF23, TAF25)	0.621 kb/206aa, Chr. IV, inviable	involved in RNA polymerase II transcription initiation and in chromatin modification
TAF12(aka TAF61, TAF68)	1.620 kb/539aa, Chr. IV, inviable	involved in RNA polymerase II transcription initiation and in chromatin modification, H2A analogue

Tra1 subunit	size, chromosome, null p-type	notes
Tra1	11.235 kb/3744aa, Chr. VIII, inviable	activates acidic activators such as Gal4p, similar to human TR-AP, which is involved in oncogenic processes, NuA4 histone acetyltransferase subunit

SAGA-associated factors involved in SAGA histone acetyltransferase complex

other subunits	size, chromosome, null p-type	notes
Sgf73	1.974 kb/657aa, Chr. VII , viable	helps form preinitiation complex
Sgf29	0.779 kb/259aa, Chr. III, viable	mutants sensitive to base pH
Sgf11	0.3 kb/99aa, Chr.XVI, viable	helps Ubp8p associate with SAGA and involved in H2B deubiquitylation
Ubp8	1.416 kb/471aa, Chr. XIII, viable	protease that helps deubiquitylate H2B
Sus1	gene with intron, Chr. II, viable	involved in mRNA export coupled transcription activation

Forward Primer: 5' aaaagtcttcagttaactcaggttcgtattctacattagATGTCGAAAGCTACATATAA 3' Reverse Primer: 5' cttcgaaaggaatagtagcggaaaagcttcttctacgcaTTAGTTTTGCTGGCCGCATC 3'