User:Emzodls/20109

20.109 Spring 2007

Last Name

de los Santos

First Name

Emmanuel Lorenzo

Nickname

Emzo

Course/Minor

20

Year of Graduation

2009

Email

emzodls AT mit DOT edu

Subjects this Term

20.109
7.05
6.001
6.041
21F.101

Module 1 : M13 Genome Re-engineering

M13 Redesign Ideas

Gene	function	design ideas
I	assembly	modify genes I, IV and XI to allow other useful proteins to attach to the complex (more flexibility in phage modification
II	replication of DNA + strand	add some kind of control to deactivate protein (e.g. sensitivity to heat or a chemical stimuli), this allows better control of the phages
III	phage tail protein	add myc epitope, or another marker that can be used for experimentation
IV	assembly	(see modification for I)
V	binds ssDNA	add tag to track changes, change structure to allow more DNA to fit in the phage
VI	phage tail protein	bind it tighter to phage body, modify protein to allow more modifications to p3
VII	phage head protein	(see p6), make protein 9 more flexible to modifications
VIII	phage coat protein	add myc or another tag (x-ray sensitive, UV sensitive, flourescent)
IX	phage head protein	tag, allow p9 to attach to different kinds of proteins including p3 (makes virus chains)
X	DNA replication	add another way to control phage propagation
XI	assembly	(see modification for I)

M13.1 Section Design

Modification/Limitation	Description
G10 ORF and G5 promoter	removed overlap between the two sections of code, changed some of the bases in the wobble position (silent mutations) in the ORF randomly to prevent recombination from homologous sequences, added overlapping KpnI and EagI sites.
G3 promoter and G8 ORF	similar to g10 ORF, g5 promoter modification, added overlapping SpeI and BclI restriction sites.
G7 rbs and G5 ORF	like above, added overlapping ApaI & AvrII restriction sites.
(G8 promoter and G9 rbs) and G7 ORF	like above, added overlapping BspEI & BglII restriction sites.
(G8 promoter and G9 rbs) and G7 ORF	like above, added overlapping BspEI & BglII restriction sites.
G8 RBS and G9 ORF, G9 ORF and G8 ORF	like above, added overlapping NcoI & NarI restriction sites.
additional g8 copy	added an aditional g8 copy (promoter, rbs and ORF) this gives two copies of the gene to work with, hopefully each copy is equally expressed in the phage coat, later on, these two copies could be modified to have affinities for two different types of metal allowing us to get an almost equal ratio of materials on the surface of the phage coat. Added overlapping KasI & BsiWI to the ends of this extra gene to make it a relatively reversible transformation.
G8 promoter and G9 rbs limitation	i decided not to separate the two as g9 does not have its own promoter and i inferred that the g8 promoter might also function as g9's promoter

Most of the modifications I made to this section involved removing the overlaps between the gene sections when possible. I did this by first copying the overlapping sequence. I then changed some of the bases at the wobble positions in the open reading frame sections that I duplicated, I made sure that the codon still codes for the same base, this way we can safeguard against recombination. My contribution to the M13 refactoring is the idea of adding another copy of gene 8. Im hoping that both copies are expressed on the cell surface. If this is the case we can modify each copy of the gene independently and make them attract different kinds of materials. This way we can get two different types of material on a single phage.

My Redesigned Phage Section

Module 3 : Expression Engineering

SAGA Protein Subunit Descriptions

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	adds structural integrity to SAGA Complex, when deleted mutants show reduced fitness in rich media and decreased metabolite accumulation, growth defect with non-fermentable carbon source
Ada2 (aka SWI8)	1.305 kb/434aa, Chr. IV, viable	transcription coactivator, when deleted mutants show reduced fitness in rich media, increased metabolite accumulation (glycogen), decreased drug resistance
Ada3(aka NGG1, SWI7)	2.109 kb/702aa, Chr. IV, viable	transcriptional regulator, decreased drug resistance, reduced fitness in rich media
Gcn5 (aka ADA4, SWI9)	1.32 kb/439aa, Chr. VII, viable	Histone acetyltransferase, catalytic subunit of ADA and SAGA, when deleted showed reduced fitness in rich media
Ada5 (aka SPT20)	1.815 kb/604aa, Chr. XV, viable	subunit of SAGA transcriptional regulatory complex, adds structural integrity, when deleted mutants show decreased drug resistance and and decreased metabolite accumulation

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, when deleted show reduced fitness in rich media, show decreased resistance to wortmannin and increased resistance to rapamycin, decreased metabolite accumulation
Spt7(aka GIT2)	3.999 kb/1332aa, Chr. II, viable	involved in proper assembly of SAGA complex, increased drug resistance to rapamycin, decreased to wortmannin
Spt8	1.809 kb/602aa, Chr. XII, viable	required for SAGA-mediated inhibition at some promoters, same thing about drug resistance, decreased metabolite accumulation
Spt20 (aka Ada5)	1.815 kb/604aa, Chr. XV, viable	adds structural integrity to SAGA Complex, same thing about drug resistance, decreased metabolite accumulation

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, dead
TAF6 (aka TAF60)	1.551 kb/516aa, Chr. VII, inviable	same as top, dead
TAF9 (aka TAF17)	0.474 kb/157aa, Chr. XIII, inviable	same
TAF10 (aka TAF23, TAF25)	0.621 kb/206aa, Chr. IV, inviable	same
TAF12(aka TAF61, TAF68)	1.620 kb/539aa, Chr. IV, inviable	same

Tra1 subunit	size, chromosome, null p-type	notes
Tra1	11.235 kb/3744aa, Chr. VIII, inviable	interacts with acidic activators to initiate transcription, dead

other subunits	size, chromosome, null p-type	notes
Sgf73	1.974 kb/657aa, Chr. VII , viable	involved in formation of the preinitiation complex assembly at promoters, decreased metabolite accumulation, reduced fitness in rich media
Sgf29	0.779 kb/259aa, Chr. III, viable	function unknown, pH sensitive after 8 generations, reduced fitness in rich media
Sgf11	0.3 kb/99aa, Chr.XVI, viable	regulates transcription of a subset of SAGA-regulated genes, required for the Ubp8p association with SAGA and for H2B deubiquitylation, when deleted showed reduced fitness in rich media
Ubp8	1.416 kb/471aa, Chr. XIII, viable	Ubiquitin-specific protease that is a component of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) acetylation complex; required for SAGA-mediated deubiquitination of histone H2B, reduced fitness in rich media
Sus1	gene with intron, Chr. II, viable	Protein involved in mRNA export coupled transcription activation

Module 4 : Biomaterials Engineering

Research Proposal