User:Nzimm

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

• 20.109
• 7.05
• 14.01
• 21M.301

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