puf: the magic rna binding protein -...
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PUF: the magic RNA binding protein
University of Illinois iGEM Angela Chen Dept. of Chemical Engineering
Anthony Chau Dept. of Molecular and Cellular Biology
Uros Kuzmanovic Dept. of Molecular and Cellular Biology
Adi Malik Dept. of Bioengineering
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Overview
• Research Motivation
• Methods and Design
• Results – What did we learn?
– Why useful?
– What practical applications?
• Future Work
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Programmable Binding Specificity
NYxxQ: recognize U SYxxR: recognize C CRxxQ or SRxxQ: recognize A SYxxE: recognize G
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Our project takes advantage of PUF’s specific binding and cutting to create a cellular tool
Bio-conveyer belt
RNA restriction enzyme
Localization
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Our project takes advantage of PUF’s specific binding and cutting to create a cellular tool
PIN (non-specific endonuclease)
PUF (site specific RNA binding protein)
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Construct Design
PUF-PIN
Reporter
mPUF-PIN
Reporter
Promoter Ribosome Binding Site Terminator
UGUAUAUA UUGAUAUA
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Fluorescence Reporter PUF-PIN mPUF-PIN
Control binding site On On
PUF binding site Off On
mPUF binding site On Off
Theoretical Data
Part Characterization
Sample Content
1 DH5a
2 Control Plasmid
3 YFP-Control Binding Site
4 YFP-PUF Binding Site
5 YFP-Control Binding Site
+ pBAD30
6 YFP-PUF Binding Site +
pBAD30
7 YFP-PUF Binding Site +
PUF-pBAD30
8 YFP-Control Binding Site
+ PUF-pBAD30
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1 2 3 4 5 6 7 8
No
rmal
ize
d F
luo
resc
en
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Sample
YFP Fluorescence Test
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Sample Content
1 DH5a
2 Control Plasmid
3 mcherry-Control Binding
Site
4 mcherry-Control Binding
Site + PUF-pBAD30
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0
1000
2000
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7000
No
rmal
ize
d F
luo
resc
en
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mCherry Fluorescence Test
1 2 3 4
Part Characterization
Conclusions
• Data suggest PUF-PIN RNA scissors has the ability to silence genes
• Further testing will provide better understanding of PUF-PIN’s functionality in E. coli
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Why Scaffold?
http://www.tbiomed.com/content/figures/1742-4682-2-33-1-l.jpg 19
Scaffold
Organization of Intracellular Reactions with Rationally Designed RNA AssembliesCamille J. Delebecque, Ariel B. Lindner, Pamela A. Silver, and Faisal A. Aldaye Science 22 July 2011: 333 (6041), 470-474.Published online 23 June 2011 [DOI:10.1126/science.1206938]
RNA Scaffold Design
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d0 Scaffold DNA Sequence: Modified d0 Sequence with PUF Binding Sites: IDT miniGene Scaffold DNA Sequence:
Wild Type PUF RNA Binding Site: UGUAUAUA (6-2/7-2) PUF RNA Binding Site: UUGAUAUA
Organization of Intracellular Reactions with Rationally Designed RNA AssembliesCamille J. Delebecque, Ariel B. Lindner, Pamela A. Silver, and Faisal A. Aldaye Science 22 July 2011: 333 (6041), 470-474.Published online 23 June 2011 [DOI:10.1126/science.1206938]
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RNA Scaffold Design http://rna.informatik.uni-freiburg.de:8080/
http://rna.informatik.uni-freiburg.de:8080/ 22
RNA Scaffold Design
http://rna.informatik.uni-freiburg.de:8080/ 23
RNA Scaffold Design
http://rna.informatik.uni-freiburg.de:8080/ 24
RNA Scaffold Design
http://rna.informatik.uni-freiburg.de:8080/ 25
RNA Scaffold Synthesis
1 2 3 4
Lanes: 1/4 – NEB Low Range ssRNA Ladder 2 – In-Vitro Transcription Run #1 3 – In-Vitro Transcription Run #2
Quantification
IV-T #1 IV-T #2
Concentration (ng/uL) 9.77 28.59
Concentration (uM) 0.22772 0.66639
A260/A280 1.538 1.444
150bp
300bp
80bp
In-Vitro Transcription with MEGAscript® T7 Kit (Invitrogen) 26
WT PUF-PIN Protein Purification
75kDa
50kDa
37kDa
25kDa
Lanes: 1 – Bio-Rad Precision Plus Protein Kaleidoscope Ladder 2 – Flow through 3 – 20 mM Imidazole 4 – 40 mM Imidazole 5 – 60 mM Imidazole 6 – 80 mM Imidazole 7 – 100 mM Imidazole 8-13 – 500 mM Imidazole elution fractions
1 2 3 4 5 6 7 8 9 10 11 12 13
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(6-2/7-2) mPUF-PIN Protein Purification
75kDa
50kDa
37kDa
25kDa
Lanes: 1 – Bio-Rad Precision Plus Protein Kaleidoscope Ladder 2 – Flow through 3 – 20 mM Imidazole 4 – 40 mM Imidazole 5 – 60 mM Imidazole 6 – 80 mM Imidazole 7 – 100 mM Imidazole 8-14 – 500 mM Imidazole elution fractions
1 2 3 4 5 6 7 8 9 10 11 12 13 14
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Future Directions
Organization of Intracellular Reactions with Rationally Designed RNA AssembliesCamille J. Delebecque, Ariel B. Lindner, Pamela A. Silver, and Faisal A. Aldaye Science 22 July 2011: 333 (6041), 470-474.Published online 23 June 2011 [DOI:10.1126/science.1206938] http://www.bio.miami.edu/~cmallery/150/memb/c11x11enzyme-cascade.jpg
Scaffold
Proving split-GFP flourescence Producing an enzyme conveyor belt for piceatannol production
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Characterization of the Petrobrick: Microbial Alkane Production Pathway
LARGE GRAPHIC
*Specifically tested for production of C15 alkanes
University of Washington. (2011). Petrobrick Components [Image]
University of Washington (2011). Fatty Acid Biosynthesis Pathway. [Image]
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Characterization of the Petrobrick: Results Summary
Maximum yield: 190 mg/L
0.00
50.00
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1 2 3 4
Alk
ane
con
cen
trat
ion
(m
g/L)
Sample
Concentration Yield (mg/L)
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Synthetic biology researchers and iGEM teams must think beyond science to economic and social consequences.
?
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1970 2003 2008 2010
UIUC iGEM has created a human practices project that allows us as researchers and the public to think deeply about synthetic biology
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UIUC iGEM has spoken to all age groups about the science and safety behind synthetic biology.
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2012 Engineering Open House UIUC Institute of Genomic Biology
To encourage long lasting conversations and education PowerPoint and video presentations on synbio were created.
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Special Thanks (alphabetical order)
Professor Kaustubh Bhalerao Dept. Agric and Bio Engineering at UIUC Professor Joe Bradley Dept. Industrial and Enterprise Systems Engineering Professor Yong-Su Jin Dept. Bioengineering at UIUC Professor Christopher Rao Dept. Chemical Engineering UIUC Professor ZeFeng Wang Dept. Pharmacology at University of North Carolina
Ahmet Badur Dept. Chemical Engineering UIUC Kori Dunn Dept. Chemical Engineering at UIUC Dr. Brad Evans Institute of Genomic Biology Courtney Fuentes-Evans Institute of Genomic Biology Todd Freestone Dept. Chemical Engineering UIUC Melissa McKillip Institute of Genomic Biology