iGEM Tsinghua-D Team

2012/10/5

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Contents

Introduction

Project

Application

Human practice

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Temperature-response Regulatory RNA –

RNA Thermometer (RNAT)

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Brief Introduction to RNAT

• First reported in 1989 in lambda phage

• Conservation : From bacteria to mammal

• Functional principle: Hairpin structure

1. Altuvia S, Kornitzer D and Teff, D, Oppenheim. Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation. Journal of Molecular Biology. 1989, Vol. 210, 265–280. 2. Shamovsky I， Ivannikov M, Kandel ES, Gershon D and Nudler E. RNA-mediated response to heat shock in mammalian cells. Nature. 2006, Vol. 440, 556–560. 3. Narberhaus F . Translational control of bacterial heat shock and virulence genes by temperature-sensing mRNAs. RNA biology. 2010, 7, 84–89.

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Previous Research

• Secondary structure prediction, 1980s

• Multi-stable RNA design accomplished in 2001 – prototype

• Man-made RNAT accomplished in 2008 – non-predefined regulation temperature

1. C Flamm, I L Hofacker, S Maurer-Stroh, et al. Design of multistable RNA molecules. RNA, 2001, Vol. 7, 254–265. 2. Juliane Neupert, Daniel Karcher and Ralph Bock. Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli. Nucleic Acids Research, 2008, Vol. 36, 19

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Our Contribution

• RNAThermo – design RNAT according to

regulation temperature

• Wet-lab Verification – structurally and

functionally

• Application – in research and industry

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Project Outline

In silico design of RNATs

Structural Verification

Functional Verification

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RNAThermo Design

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Physical Principles

1. David H. Mathews. Revolutions in RNA secondary structure prediction. J. Mol. Biol., 2006, Vol. 359, 526–532 2. David H Mathews and Douglas H Turner. Prediction of RNA secondary structure by free energy minimization. Current Opinion in Structural Biology, 2006, Vol. 16, 270–278 3. J. S. McCASKlLL. The Equilibrium partition function and base pair binding probabilities for RNA secondary structure. Biopolymers, 1990, Vol. 29,1105-1119

Secondary structure prediction

Free energy minimization

Partition function

Structure with

minimal free

energy

Probability of

each structures’

appearance

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Algorithm

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Algorithm

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Algorithm

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Algorithm

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An Example

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Download of RNAThermo

• For academic users, we provide download service.

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Verification

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• Structural

• Functional

Structural Verification

Elizabeth E. Regulski and Ronald R. Breaker. In-Line probing analysis of riboswitches. Methods in Molecular Biology, 2008, Vol. 419, 53-67 18

Functional Verification GFP RNAT

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Results

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Future Application

Industry

• Lysate engineered microorganism to extract the products

• Avoid toxic products expression during cell accumulation

• Medicine for specific disease, like gastric ulcer

Fundamental

research

• Temperature detector

• Switch on/off the gene to be studied at particular period

• Align several RNATs to accomplish accurate regulation

• Communication between cells : domino effect

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Future Application: industry

Lysate engineered microorganism to

extract the products

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RNAT Lysozyme

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Future Application: Industry

Avoid toxic products expression during

cell accumulation

Medicine for specific disease, like

gastric ulcer

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RNAT Toxic Product

RNAT medicine

Future Application :

Fundamental research

Temperature detector

Switch on/off the gene to be studied at

particular period

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RNAT GFP

RNAT Developmental Gene

Future Application :

Fundamental research • Align several RNATs to accomplish

accurate regulation

• Communication between cells : domino

effect

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RNAT Gene 1

RNAT Gene 2

Future Application

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RNAT

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Human Practice

Freshmen workshop

Open house

Goal: more undergraduates

experience laboratory research

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Freshmen Workshop

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Open House

Well organized

Brief Introduction in iGEM

Epoch-making events of synthetic biology

Free talk and Q&A

Laboratory visit

Widely Influential

Students from various departments

Many are extremely attracted !

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Open house

Open house

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Open house

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Open house

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Open house

Open house

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Open house

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What did we do?

In silico design of RNATs

Functional Verification

Structural Verification

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Our Team

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• Prof. CHEN Guoqiang, Prof. SUN Zhirong and Prof. DAI Junbiao for their guidance of the project.

• Prof. Tom Kellie for his revision of the PPT and the report.

• Dr. YIN Ping and Dr. QU Peng for their help in the RNA experiments.

• FU Xiaozhi and LI Teng for their help in the molecular biology experiments.

• LI Jiatong for organizing the human practice.

Acknowledgement

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Thanks for your attention!

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