User:Nzimm

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Neil Zimmerman

Bio

  • MIT Class of 2009
  • Major: Biological Engineering
  • Minors: Management and Music
  • Varsity Soccer
  • Wind Ensemble (Clarinet)

20.109

M13K07 Genome Engineering Plans:

Gene Plans
I Change # of protein copies expressed to experiment with different sized channels
II Modify residues to allow deactivation of p2 under certain conditions so that replication of + strand can be regulated
III Insert myc to allow detection with an antibody
IV Change # of protein copies expressed to experiment with different sized channels
V Add fluorescent tag to monitor levels of p5-ssDNA complex
VI Modify residues to help p3 bind more effectively to the ToIA protein on the E. coli F pilus
VII Minimize the bulk of the protein to allow more room for modifications on p9
VIII Insert myc (as in III) or another tag to serve as a “hook” for attaching constructs to M13
IX Add residues to N-terminus to present on the outside of the phage coat
X Add sensitivity to different stimulus than that of p2 in order to regulate replication of + strand in another fashion
XI Modify residues to allow proteins other than p8 to embed in the membrane and serve as the phage filament coat

M13K07 Genome Refactoring:

In refactoring the M13K07 genome between the HpaI site in gene II and the BamHI site in gene III, I separated all overlapping elements across the region by copying overlapping sequences and placing the elements adjacent to one another on the refactored sequence. As a result, the refactored region is 1,000 bp larger than the original sequence. I also added annotation for & refactored the open reading frames of genes 2, 5, 7, and 10, which are not annotated in the M1307 part in the registry. I inserted the sequence encoding the myc protein into g3. I intend to separate each element with two unique restriction sites, so that the elements can be isolated & manipulated more easily. I initially proposed to add a fluorescent tag like GFP to g5, but seeing as how GFP is nearly 3 times the size of the protein product of g5, I chose to leave g5 unaltered. Below is a table describing the overlaps that were refactored.

Overlapping elements Action
rbs g7 & g5 ORF copied overlapping sequence & placed rbs g7 adjacent to the 3' end of the g5 ORF
promoter g8 & rbs g9 copied overlapping sequence & placed rbs g9 adjacent to the 3' end of promoter g8
promoter g3 & g8 ORF copied overlapping sqeuence & placed promoter g3 adjacent to the 3' end of g8 ORF
g10 ORF, g10 rbs, g10 promoter, g5 promoter, & g2 ORF copied g2 ORF & placed adjacent to 5' end of other (non-overlapping) elements
inserted the sequence encoding the myc protein into the BamHI site of g3

Current Courses

Other

Email: nzimm (at) mit (dot) edu

SAGA subunits, S. cerevisiae

Ada subunits size,chromosome,null p-type notes
Ada1 (aka HFI1, SUP110, SRM12, GAN1) 1.467 kb/489 aa, Chr. XVI,
viable
"Histone H2A Functional Interactor"; Adapter protein involved in structural integrity of SAGA
Ada2 (aka SWI8) 1.305 kb/434aa, Chr. IV,
viable
"transcriptional ADAptor"; Transcription coactivator
Ada3(aka NGG1, SWI7) 2.109 kb/702aa, Chr. IV,
viable
Transcriptional regulator involved in glucose repression of Gal4p-regulated genes
Gcn5 (aka ADA4, SWI9) 1.32 kb/439aa, Chr. VII,
viable
"General Control Nonderepressible"; Histone acetyltransferase, acetylates N-terminal lysines on histones H2B and H3; this is the catalytic subunit
Ada5 (aka SPT20) 1.815 kb/604aa, Chr. XV,
viable
"SuPpressor of Ty"; involved in maintaining the integrity of the SAGA transcriptional regulatory complex
Spt subunits size, chromosome, null p-type notes
Spt3 1.014 kb/337aa, Chr. IV,
viable
interacts with Spt15p to activate transcription of some RNA polymerase II-dependent genes, also functions to inhibit transcription at some promoters
Spt7(aka GIT2) 3.999 kb/1332aa, Chr. II,
viable
involved in proper assembly of the complex; also present as a C-terminally truncated form in the SLIK/SALSA transcriptional regulatory complex
Spt8 1.809 kb/602aa, Chr. XII,
viable
required for SAGA-mediated inhibition at some promoters; not present in SLIK/SALSA complex
Spt20 (aka Ada5) 1.815 kb/604aa, Chr. XV,
viable
maintains integrity of complex
TAF subunits size, chromosome, null p-type notes
TAF5 (aka TAF90) 2.397 kb/798aa, Chr. II, inviable "TATA binding protein-Associated Factor"; RNA polymerase II transcription initiation and in chromatin modification (90 kDa subunit)
TAF6 (aka TAF60) 1.551 kb/516aa, Chr. VII, inviable 60 kDa subunit with same function as above; similar to Histone H4
TAF9 (aka TAF17) 0.474 kb/157aa, Chr. XIII, inviable 17 kDa subunit; similar to Histone H3
TAF10 (aka TAF23, TAF25) 0.621 kb/206aa, Chr. IV, inviable 145 kDa subunit
TAF12(aka TAF61, TAF68) 1.620 kb/539aa, Chr. IV, inviable 61/68 kDa subunit; similar to Histone H2A
Tra1 subunit size, chromosome, null p-type notes
Tra1 11.235 kb/3744aa, Chr. VIII, inviable interacts with acidic activators (e.g., Gal4p) which leads to transcription activation; similar to human TRRAP, which is a cofactor for c-Myc mediated oncogenic transformation
other subunits size, chromosome, null p-type notes
Sgf73 1.974 kb/657aa, Chr. VII ,
viable
"SaGa associated Factor"; formation of the preinitiation complex assembly at promoters (73 kDa subunit)
Sgf29 0.779 kb/259aa, Chr. III,
viable
29 kDa subunit of SAGA histone acetyltransferase complex
Sgf11 0.3 kb/99aa, Chr.XVI,
viable
Integral subunit of SAGA histone acetyltransferase complex, regulates transcription of a subset of SAGA-regulated genes, required for the Ubp8p association with SAGA and for H2B deubiquitylation (11 kDa)
Ubp8 1.416 kb/471aa, Chr. XIII,
viable
Ubiquitin-specific protease - required for SAGA-mediated deubiquitination of histone H2B
Sus1 gene with intron, Chr. II,
viable
"Sl gene Upstream of ySa1"; involved in mRNA export and interacts with the SAGA histone acetylase complex for transcription activation; component of the SAGA histone acetylase complex; high concentrations at nuclear pores

20.109:Module 4 Research Proposal

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