User:Msoh: Difference between revisions
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| subunit involved in structural integrity | | subunit involved in structural integrity | ||
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TATA binding protein-Associated Factors | |||
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! TAF subunits | ! TAF subunits | ||
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| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF5 TAF5] (aka TAF90) | | [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF5 TAF5] (aka TAF90) | ||
| 2.397 kb/798aa, Chr. II, <font color = red>inviable</font color> | | 2.397 kb/798aa, Chr. II, <font color = red>inviable</font color> | ||
| | | involved in RNA polymerase II transcription initiation and in chromatin modification, mutants inviable | ||
|- | |- | ||
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF6 TAF6] (aka TAF60) | | [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF6 TAF6] (aka TAF60) |
Revision as of 11:13, 3 April 2007
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 resistnace |
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, mutants inviable |
TAF6 (aka TAF60) | 1.551 kb/516aa, Chr. VII, inviable | |
TAF9 (aka TAF17) | 0.474 kb/157aa, Chr. XIII, inviable | |
TAF10 (aka TAF23, TAF25) | 0.621 kb/206aa, Chr. IV, inviable | |
TAF12(aka TAF61, TAF68) | 1.620 kb/539aa, Chr. IV, inviable |
Tra1 subunit | size, chromosome, null p-type | notes |
---|---|---|
Tra1 | 11.235 kb/3744aa, Chr. VIII, inviable |
other subunits | size, chromosome, null p-type | notes |
---|---|---|
Sgf73 | 1.974 kb/657aa, Chr. VII , viable |
|
Sgf29 | 0.779 kb/259aa, Chr. III, viable |
|
Sgf11 | 0.3 kb/99aa, Chr.XVI, viable |
|
Ubp8 | 1.416 kb/471aa, Chr. XIII, viable |
|
Sus1 | gene with intron, Chr. II, viable |