User:Msoh

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Contents

Michael Oh

Class of 2009

Email: 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'

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