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==20.109 Spring 2007==
== Emmanuel Lorenzo (Emzo) de los Santos ==
 
Graduate Student in [http://www.be.caltech.edu/ Bioengineering] at Caltech <br>
===Last Name===
Co-advised by [http://mayo.caltech.edu Stephen Mayo] and [http://www.cds.caltech.edu/~murray/wiki/Main_Page Richard Murray] <br>
de los Santos
S.B. 2009 from [http://web.mit.edu MIT] in [http://web.mit.edu/be Biological Engineering]<br>
 
emzodls "at" caltech "dot" edu
===First Name===
Emmanuel Lorenzo
===Nickname===
Emzo
===Course/Minor===
20
===Year of Graduation===
2009
===Email===
emzodls AT mit DOT edu
 
===Subjects this Term===
20.109<br>
7.05<br>
6.001<br>
6.041<br>
21F.101
 
==Module 1 : M13 Genome Re-engineering==
 
===M13 Redesign Ideas===
{| Border="2"
! 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===
 
{| Border="2"
! 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.
 
[http://parts.mit.edu/registry/index.php/Part:BBa_M31512 My Redesigned Phage Section]
 
 
 
===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<br>
7.05<br>
6.001<br>
6.041<br>
21F.101
 
==Module 3 : Expression Engineering==
 
===SAGA Protein Subunit Descriptions===
 
====SAGA subunits, <i> S. cerevisiae </i>====
 
{| border="1" 
! Ada subunits 
! size,chromosome,null p-type
! notes
|-   
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA1 Ada1] (aka HFI1, SUP110, SRM12, GAN1)
| 1.467 kb/489 aa, Chr. XVI, <br>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
|-   
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA2 Ada2] (aka SWI8) 
| 1.305 kb/434aa, Chr. IV, <br>viable
| transcription coactivator, when deleted mutants show reduced fitness in rich media, increased metabolite accumulation (glycogen), decreased drug resistance
|-   
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA3 Ada3](aka NGG1, SWI7) 
| 2.109 kb/702aa, Chr. IV, <br>viable 
| transcriptional regulator, decreased drug resistance, reduced fitness in rich media
|-  
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=GCN5 Gcn5] (aka ADA4, SWI9) 
| 1.32 kb/439aa, Chr. VII, <br>viable
| Histone acetyltransferase, catalytic subunit of ADA and SAGA, when deleted showed reduced fitness in rich media
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Ada5 Ada5] (aka SPT20)
| 1.815 kb/604aa, Chr. XV, <br>viable
| subunit of SAGA transcriptional regulatory complex, adds structural integrity, when deleted mutants show decreased drug resistance and and decreased metabolite accumulation
|}
{| border="1" 
! Spt subunits
! size, chromosome, null p-type
! notes 
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=spt3 Spt3]
| 1.014 kb/337aa, Chr. IV, <br> 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
|-   
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Spt7 Spt7](aka GIT2) 
| 3.999 kb/1332aa, Chr. II, <br> viable
| involved in proper assembly of SAGA complex, increased drug resistance to rapamycin, decreased to wortmannin
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Spt8 Spt8]
| 1.809 kb/602aa, Chr. XII, <br> viable
| required for SAGA-mediated inhibition at some promoters, same thing about drug resistance, decreased metabolite accumulation
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=SPT20 Spt20] (aka Ada5) 
| 1.815 kb/604aa, Chr. XV, <br> viable
| adds structural integrity to SAGA Complex, same thing about drug resistance, decreased metabolite accumulation
|- 
|}
{| border="1"
! TAF subunits
! size, chromosome, null p-type
! notes
|- 
| [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>
| involved in RNA polymerase II transcription initiation and in chromatin modification, dead
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF6 TAF6] (aka TAF60) 
| 1.551 kb/516aa, Chr. VII, <font color = red>inviable</font color>
| same as top, dead
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF9 TAF9] (aka TAF17) 
| 0.474 kb/157aa, Chr. XIII, <font color = red>inviable</font color>
| same
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF10 TAF10] (aka TAF23, TAF25)
| 0.621 kb/206aa, Chr. IV, <font color = red>inviable</font color>
| same
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TAF12 TAF12](aka TAF61, TAF68)
| 1.620 kb/539aa, Chr. IV, <font color = red>inviable</font color>
| same
|}
{| border="1"  
! Tra1 subunit 
! size, chromosome, null p-type
! notes
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=TRA1 Tra1] 
| 11.235 kb/3744aa, Chr. VIII, <font color = red>inviable</font color>
| interacts with acidic activators to initiate transcription, dead
|}
{| border="1"
! other subunits 
! size, chromosome, null p-type
! notes
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf73 Sgf73] 
| 1.974 kb/657aa, Chr. VII , <br> viable
| involved in formation of the preinitiation complex assembly at promoters, decreased metabolite accumulation, reduced fitness in rich media
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf29 Sgf29] 
| 0.779 kb/259aa, Chr. III, <br> viable
| function unknown, pH sensitive after 8 generations, reduced fitness in rich media
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf11 Sgf11] 
| 0.3 kb/99aa, Chr.XVI, <br> 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
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ubp8 Ubp8] 
| 1.416 kb/471aa, Chr. XIII, <br> 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
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Sus1 Sus1] 
| gene with intron, Chr. II, <br> viable 
| Protein involved in mRNA export coupled transcription activation
|}

Latest revision as of 17:58, 8 November 2010

Emmanuel Lorenzo (Emzo) de los Santos

Graduate Student in Bioengineering at Caltech
Co-advised by Stephen Mayo and Richard Murray
S.B. 2009 from MIT in Biological Engineering
emzodls "at" caltech "dot" edu

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