IRESistible: Novel Parts for Use in S. cerevisiaeweb.utk.edu/~ctrinh/UTK_iGEM2012 oral.pdf ·...
Transcript of IRESistible: Novel Parts for Use in S. cerevisiaeweb.utk.edu/~ctrinh/UTK_iGEM2012 oral.pdf ·...
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IRESistible: Novel Parts for Use in S. cerevisiae
UTK-Knoxville iGEM team Morgan Baltz, Katie Lutes, and Akshitha Yarrabothula
October 13, 2012
July
Get it? … IRESis-ble LOL
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Outline
§ Who we are § Motivation § What is an IRES – How does IRES mediated translation differ
from cap dependent translation § Applications § Completed work § Proposed work § Conclusions
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Who we are The University of Tennessee, Knoxville
Go Big Orange!!
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Who we are § We are UT’s inaugural iGEM team § Morgan, Katie, and Akshitha were the
core members
Genesis Minter Brandon Wilbanks
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Who we are
Michael Wierzbicki works with E. coli Adam Thompson works with S. cerevisiae
Dr. Cong Trinh was our primary advisor Dr. Dan Close works with IRESs
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Motivation
Promoter Gene1 Terminator Promoter Gene 2 Terminator
Promoter Gene1 Gene 2 Terminator
In prokaryotes, mul-ple genes can be expressed under the control of the same promoter.
In eukaryotes, each gene must be expressed under the control of its own promoter.
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What is an IRES?
Internal Ribosomal Entry Site
July
Hey! That looks like me! Tumban et al. Journal of Nega-ve Results in BioMedicine 2009 8:4 doi:10.1186/1477-‐5751-‐8-‐4
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What is an IRES?
>2?78$*:@)AB4:$ (2;4525$$
C9;?2B4:78$0;7?D7B4:$ EFG4H27$
!"##$%&'"(($
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Motivation § Why – The Parts Registry has no IRESs – IRESs included in other parts are poorly
documented § Goals – Introduce IRESs to the Synthetic Biology
community because IRESs: • Allow for protein expression under one promoter • Drastically reduce the size of the construct • Reduce likelihood of recombination
– Create a method of standardizing IRES strength
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Traditional mechanism:
AUG$
AAAAAAAAAAAAAAAAAAAA$
PABP$
7MGPPPG$
eIF4E$
eIF4G$
eIF3$ eIF4A$
40S$Ribosomal$$Subunit$
Cap dependent translation initiation
3’
5’
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!"#$
!!!!!!!!!!!!!!!!!!!!
$
%!&%$
'(#%%%#$
)*+,-$
)*+,#$
*<!+$)*+.$
)*+,!$
,/0$123454678$$0939:2;$
=1+$ !"#$
IRES mechanism: Cap independent translation initiation
3’
5’ eIF4A
ITAF
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Application § How can synthetic biologists use
IRESs – reporter genes • example: pIRES commercial vector • example: AIDS kittens
July
Those cats are almost as IRESis-ble as we are!
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Application Estrogenic Hormone Biosensor
Schematic representation of S. cerevisiae BLYES. Estrogenic compounds cross the cell membrane and bind to the estrogen receptor.
Sanseverino J et al. Appl. Environ. Microbiol. 2005;71:4455-4460
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Completed work
Name Description Length
BBa_K813000 YAP1 -‐ Yeast Genomic IRES 164
BBa_K813001 URE2 -‐ Yeast Genomic IRES 167
BBa_K813002 HAP4 -‐ Yeast Genomic IRES 270
BBa_K813003 pSAP -‐ Yeast Genomic IRES 528
BBa_K813004 p150 -‐ Yeast Genomic IRES 348
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Completed work
PART ORIGIN Backbone (BBa_J63010) 2012 Kit, Plate 1, Well 1C
ADH1 (BBa_J63005) 2012 Kit, Plate 1, Well 1C
mOrange (BBa_E2050) 2012 Kit, Plate 2, Well 13N
GFP (BBa_I13522) 2011 Kit, Plate 2, Well 8A
cyc1 Trinh Lab
All IRESs S. cerevisiae genomic DNA
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Completed work
Completed IRES Characteriza=on Construct
9400 bp
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Comparison
Construct with IRES
9,400 bp
Construct without IRES 10,559 bp
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Proposed work
mOrange
GFP
ADH1 mOrange Cyc
mOrange GF
P
ADH1 GFP Cyc
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Proposed work
mOrange
GFP
ADH1 mOrange IRES GFP Cyc
mOrange
GFP
Rela=vely weak IRES Rela=vely strong IRES
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Proposed work
IRES GFP Cyc
mOrange
GFP
July
Hey I don’t work that way!
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Conclusions
Problems § Antibiotic resistance § ADH1 promoter § Limited experience with S. cerevisiae § Small team § Limited resources
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Conclusions
What we learned § IRESs § Yeast techniques § BioBrick § Wiki § Flow cytometry § Research project management
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Acknowledgments § The University of Tennessee, Knoxville College of
Engineering § UT-ORNL Joint Institute for Biological Studies § The University of Tennessee, Knoxville Office of
Research § IDT § NEB § The Parts Registry § iGEM § Duquesne University § Dr. Cong Trinh
July
Thanks!