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 RNA Binding Protein?

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The pumilio protein consists of 8 modular repeats that specifically bind to nucleotide bases

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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)

METHODS & DESIGN “Our Approach”

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Construct Design

PUF-PIN

mPUF-PIN

Promoter Ribosome Binding Site Terminator

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Construct Design

PUF-PIN

Reporter

mPUF-PIN

Reporter

Promoter Ribosome Binding Site Terminator

UGUAUAUA UUGAUAUA

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Construct Design

PUF-PIN mPUF-PIN

Promoter Ribosome Binding Site Terminator

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Reporter

Construct Design

PUF-PIN

Promoter Ribosome Binding Site Terminator

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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

RESULTS “What did we learn? Why Important?”

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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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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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mCherry Fluorescence Test

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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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Toward Practical Applications “Multiple Applications and Opportunities”

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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

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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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RNA Scaffold Endonuclease Assay

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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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University of Washington Petrobrick Characterization

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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

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RESEARCH TEAM OUTREACH “Future Growth in Synthetic Biology”

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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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SUPPORT SYSTEM “Could not be successful without them”

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University of Illinois at Urbana – Champaign iGEM team!

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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

Special thanks to you, our audience, and our sponsors

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