User:Alji: Difference between revisions

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==== M13 Refactoring ====
==== M13 Refactoring ====


I attempted to refactor the M13K07 phage by removing the overlapping areas of genes between the HpaI site of gene 2 and the BamHI site of gene 3. I did this by duplicating each gene (promoter, rbs, and ORF) and placing it after the ORF of the previous gene. I mutated the RBS's of each overlapping area to a silent mutation to prevent readout of the overlapping sections. There were 6 places of overlap, and I have included what changes I made. As a result, I have added 769 bp to the original section of DNA. In between each gene, I left 11 extra base pairs in order to introduce restriction sites so that each part can be manipulated individually. I want to see what others have done before attempting to introduce new restriction endonuclease sites.


{| Border="1"
{| Border="1"
! Overlapping Areas !! Modified sequence
! Overlapping Areas !! Original Sequence !! Modified sequence
|-
|-
| Gene 2 ORF and Gene 10 RBS || ||  
| Gene 2 ORF and Gene 10 RBS || At bp 478: gcatttgag || gcGtttgag
|-
|-
| Gene 10 ORF and Gene 5 RBS || ||  
| Gene 2 ORF and Gene 5 RBS || At bp 826: gcataaggt || gcGtaaggt
|-
|-
| Gene 5 ORF and Gene 7 RBS || ||  
| Gene 10 ORF and Gene 5 RBS || At bp 1286: gcataaggt || gcGtaaggt
|-
|-
| Gene 5 ORF and Gene 9 RBS || ||  
| Gene 5 ORF and Gene 7 RBS || At bp 1609: gttccggct || gtCccggct
|-
|-
| Gene 9 ORF and Gene 8 RBS ||  ||  
| Gene 7 ORF and Gene 9 RBS || At bp 1733: cgctggggg || cgAtggggg
|-
| Gene 9 ORF and Gene 8 RBS || At bp 1869: aatggaaac || aaCggaaac
|}
 
The refactored genome is [http://parts.mit.edu/registry/index.php/Part:BBa_M31332 Part BBa_M31332] on the Registry of Standard Biological Parts
 
 
====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
| 
|-   
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA2 Ada2] (aka SWI8) 
| 1.305 kb/434aa, Chr. IV, <br>viable
|
|-   
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ADA3 Ada3](aka NGG1, SWI7) 
| 2.109 kb/702aa, Chr. IV, <br>viable 
|
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=GCN5 Gcn5] (aka ADA4, SWI9) 
| 1.32 kb/439aa, Chr. VII, <br>viable
|
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Ada5 Ada5] (aka SPT20)
| 1.815 kb/604aa, Chr. XV, <br>viable
|
|}
{| 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
|
|-   
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Spt7 Spt7](aka GIT2) 
| 3.999 kb/1332aa, Chr. II, <br> viable
|
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Spt8 Spt8] 
| 1.809 kb/602aa, Chr. XII, <br> viable
|
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=SPT20 Spt20] (aka Ada5) 
| 1.815 kb/604aa, Chr. XV, <br> viable
|
|}
{| 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>
|
|-
| [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>
|
|- 
| [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>
|
|-
| [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>
|
|-
| [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>
|
|}
{| 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>
|
|}
{| 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
|
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf29 Sgf29] 
| 0.779 kb/259aa, Chr. III, <br> viable
|
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf11 Sgf11] 
| 0.3 kb/99aa, Chr.XVI, <br> viable
|
|-
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=ubp8 Ubp8] 
| 1.416 kb/471aa, Chr. XIII, <br> viable
|
|- 
| [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=Sus1 Sus1] 
| gene with intron, Chr. II, <br> viable 
|
|}
 
 
 
{| border="1" 
! Primer
! Primer Number
! Sequence
! Anneals
|- 
| ''URA3'' replacement PCR Forward Primer
| NO177
| 5' GCGACAAAATCAGAAGTAACAATTCTGGCCTTCACTCCAatgtcgaaagctacatataa
| 20 bp upstream of ''SUS1''
|-
| ''URA3'' replacement PCR Reverse Primer 
| NO178
| 5' TGTAATAATATTGGGAATTAAGGTGCATTTTCGTATCCTttagttttgctggccgcatc
| 20 bp downstream of ''SUS1''
|-
| Colony PCR Forward Primer
| NO193
| 5' AATGGTTAAGATACCAATGCCGTCTACACC
| 520 bp upstream of ''SUS1''
|- 
| Colony PCR Reverse Primer
| NO179
| 5' CTGTGCCCTCCATGGAAAAATCAGTCAAGA
| 191-220 bp (btm strand) after ''URA3'' ATG
|}
|}


The refactored genome is [] on the Registry of Standard Biological Parts
 
{| border="1" 
! Media
! Temperature
|- 
| YP Galactose
| 23 C
|-
| YP Galactose
| 30 C
|-
| YP Galactose
| 37 C
|- 
| YP Dextrose
| 23 C
|-
| YP Dextrose
| 30 C
|-
| YP Dextrose
| 37 C
|-
| YP+Rapamycin
| 30 C
|-
| YP-Lysine
| 30 C
|-
| YP
| 30 C
|-
| YP-Tryptophan
| 30 C
|}
 
 
{| border="1" 
! Transformation
! Number of Colonies
|- 
| "Plus template"
| 10
|-
| "No template"
| 3
|-
| 50 ng pRS406 DNA
| 800
|}
 
 
{| border="1"
|
! colspan="2" | Surface display of scFv fusion?
! colspan="2" | Binding to gold?
|-
|
! Glucose
! Galactose
! Glucose
! Galactose
|-
!pCT-CON
|N
|Y
|N
|Y
|-
!pAu1
|N
|Y
|N
|Y
|}
 
 
{| border="1" 
! Transformation
! Number of Colonies
|- 
| pCT-CON
| 2
|-
| Peptide library
| 3
|-
| pAu1
| 1944
|}
 
 
{| border="1" 
! Plasmid
! Altered Condition
! Number of Colonies
|- 
| pCT-CON
| 45-min. panning
| 0
|-
| pAu1
| 45-min. panning
| 0
|-
| pCT-CON
| 2X BSA
| 0
|-
| pAu1
| 2X BSA
| 0
|}

Latest revision as of 18:21, 8 May 2007

Andrew Ji

About Me

  • Year: 2009
  • Major: Biological Engineering
  • Minor: Economics
  • Email: alji AT mit DOT edu

M13K07 Genome Re-engineering ideas:

Gene Ideas
I Change size of protein to see effect of different channel sizes.
II Increase or decrease expression to optimize rate of + strand replication.
III Myc tag to monitor expression in phage and bacteria or to add other things to test initial interaction with host.
IV Change size of protein to see effect of different channel sizes.
V Add more DNA binding sites to see if more DNA can be packaged into phage.
VI Myc tag to monitor expression in phage and bacteria or to add other things to test initial interaction with host.
VII Tag protein to monitor interaction with p5/DNA complex.
VIII Change size of protein to experiment with the size of coat.
IX Tag protein to monitor interaction with p5/DNA complex.
X Increase expression to see if more phage leave the host E. coli.
XI Change size of protein to see effect of different channel sizes.


M13 Refactoring

I attempted to refactor the M13K07 phage by removing the overlapping areas of genes between the HpaI site of gene 2 and the BamHI site of gene 3. I did this by duplicating each gene (promoter, rbs, and ORF) and placing it after the ORF of the previous gene. I mutated the RBS's of each overlapping area to a silent mutation to prevent readout of the overlapping sections. There were 6 places of overlap, and I have included what changes I made. As a result, I have added 769 bp to the original section of DNA. In between each gene, I left 11 extra base pairs in order to introduce restriction sites so that each part can be manipulated individually. I want to see what others have done before attempting to introduce new restriction endonuclease sites.

Overlapping Areas Original Sequence Modified sequence
Gene 2 ORF and Gene 10 RBS At bp 478: gcatttgag gcGtttgag
Gene 2 ORF and Gene 5 RBS At bp 826: gcataaggt gcGtaaggt
Gene 10 ORF and Gene 5 RBS At bp 1286: gcataaggt gcGtaaggt
Gene 5 ORF and Gene 7 RBS At bp 1609: gttccggct gtCccggct
Gene 7 ORF and Gene 9 RBS At bp 1733: cgctggggg cgAtggggg
Gene 9 ORF and Gene 8 RBS At bp 1869: aatggaaac aaCggaaac

The refactored genome is Part BBa_M31332 on the Registry of Standard Biological Parts


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
Ada2 (aka SWI8) 1.305 kb/434aa, Chr. IV,
viable
Ada3(aka NGG1, SWI7) 2.109 kb/702aa, Chr. IV,
viable
Gcn5 (aka ADA4, SWI9) 1.32 kb/439aa, Chr. VII,
viable
Ada5 (aka SPT20) 1.815 kb/604aa, Chr. XV,
viable
Spt subunits size, chromosome, null p-type notes
Spt3 1.014 kb/337aa, Chr. IV,
viable
Spt7(aka GIT2) 3.999 kb/1332aa, Chr. II,
viable
Spt8 1.809 kb/602aa, Chr. XII,
viable
Spt20 (aka Ada5) 1.815 kb/604aa, Chr. XV,
viable
TAF subunits size, chromosome, null p-type notes
TAF5 (aka TAF90) 2.397 kb/798aa, Chr. II, 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


Primer Primer Number Sequence Anneals
URA3 replacement PCR Forward Primer NO177 5' GCGACAAAATCAGAAGTAACAATTCTGGCCTTCACTCCAatgtcgaaagctacatataa 20 bp upstream of SUS1
URA3 replacement PCR Reverse Primer NO178 5' TGTAATAATATTGGGAATTAAGGTGCATTTTCGTATCCTttagttttgctggccgcatc 20 bp downstream of SUS1
Colony PCR Forward Primer NO193 5' AATGGTTAAGATACCAATGCCGTCTACACC 520 bp upstream of SUS1
Colony PCR Reverse Primer NO179 5' CTGTGCCCTCCATGGAAAAATCAGTCAAGA 191-220 bp (btm strand) after URA3 ATG


Media Temperature
YP Galactose 23 C
YP Galactose 30 C
YP Galactose 37 C
YP Dextrose 23 C
YP Dextrose 30 C
YP Dextrose 37 C
YP+Rapamycin 30 C
YP-Lysine 30 C
YP 30 C
YP-Tryptophan 30 C


Transformation Number of Colonies
"Plus template" 10
"No template" 3
50 ng pRS406 DNA 800


Surface display of scFv fusion? Binding to gold?
Glucose Galactose Glucose Galactose
pCT-CON N Y N Y
pAu1 N Y N Y


Transformation Number of Colonies
pCT-CON 2
Peptide library 3
pAu1 1944


Plasmid Altered Condition Number of Colonies
pCT-CON 45-min. panning 0
pAu1 45-min. panning 0
pCT-CON 2X BSA 0
pAu1 2X BSA 0
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