KOREA U SEOUL INTRODUCTION TEAM: KOREA U SEOUL 2012 iGEM KOREA U - - PowerPoint PPT Presentation

KOREA U SEOUL

INTRODUCTION

TEAM: KOREA U SEOUL

2012 iGEM KOREA U SEOUL

INTRODUCTION

TEAM: KOREA U SEOUL

2012 iGEM KOREA U SEOUL

INTRODUCTION

CONTENTS

1 Project 1: Rice Gruardian

• Background information

2 Project 1: Rice Guardian

• Design and Results

3 Project 2: Biological Logic Gate 4 Human Practice

PROJECT 1: RICE GUARDIAN

Why Do We Focus on Bacterial Leaf Blight (BLB)?

Rice: feeding the billions

• >90% of total rice produced

in Asia

• Most important crop for

human

– providing 30~85 % of total calories consumed by Asian people – 8% of food energy for 1 billion people over the world (except Asia)

Bacterial leaf blight (BLB)

• Caused by Xanthomonas
• One of the most serious

disease of rice

http://eprints.hec.gov.pk/2349/1/2204.htm http://books.irri.org/

PROJECT 1: RICE GUARDIAN

2012 iGEM KOREA U SEOUL

Xanthomonas oryzae and Ax21

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

Rice

Bacterial Leaf Blight

Xanthomonas oryzae Ax21

Sang-Wook Han, Pamela C. Ronald et al. 2011. Small protein-mediated quorum sensing in a gram-negative

• bacterium. PLoS ONE. 6:12

Xanthomonas oryzae and Ax21

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

RICE GUARDIAN Xanthomonas oryzae

CLEAR Xanthomonas

Ax21

Sang-Wook Han, Pamela C. Ronald et al. 2011. Small protein-mediated quorum sensing in a gram-negative

• bacterium. PLoS ONE. 6:12

How Do We Control BLB (Xanthomonas oryzae)

• Quorum Sensing in Xanthomonas
• Signal

–Ax21 (protein)

• Receiver

– raxH gene

• Regulator

– raxR gene and its promoter

Sang-Wook Han, Sang-Won Lee and Pamela C Ronald. 2011. Secretion, modification, and regulation of Ax21, Current Opinion in Microbiology. 14:62–67

PROJECT 1: RICE GUARDIAN

2012 iGEM KOREA U SEOUL

RICE GUARDIAN: Part Design

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

Ax21 Sensing bacteria

RaxR/RaxH: a two component regulatory system in Xanthomonas which works as a receptor for Ax21.

BBa_J23100

(constitutive promoter)

mRFP raxR (colR) raxH (colS) BBa_K739003

(prax)

BBa_K739000

RICE GUARDIAN: Part Design

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN RaxH Ax21 RaxR

BBa_J23100

(constitutive promoter)

mRFP raxR (colR) raxH (colS) BBa_K739003

(prax)

Ax21 Sensing bacteria

• Problem
• Xanthomonas is pathogenic

 solution: Overexpressing Ax21 inside E. Coli

RICE GUARDIAN: Part Design

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

pAT: a vector designed to display heterologous proteins

• n cell-surface of E. coli

pBAD

BBa_K739004 (ax-21)

* T domain : Translocator domain

T domain

Ax21 Displaying Bacteria

Heok-Jin Ko, In-Geol Choi et al. 2012. Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes in Escherichia coli with a Novel Ligation Independent Cloning Vector Based on the Autotransporter YfaL. AEM. 10.1128

 Problem: We could not attain Ax21 in a soluble form  solution: DISPLAYING Ax21 on cell membrane

RICE GUARDIAN PROJECT: 4 EXPERIMENTS

Experiment 1:

Cloning: PCR from gDNA + 3A assembly

Experiment 2:

SDS-PAGE analysis of Ax21

Experiment 3:

Fluorescence and cell density of co-cultured media

Experiment 4:

Co-culture: growth curve of each cell carrying different plasmid

RICE GUARDIAN PROJECT: Exp. 1

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

• Cloning: PCR from gDNA + 3A assembly

Bba_J23100

(constitutive promoter)

raxR (colR) raxH (colS)

BBa_K739000

mRFP

prax

BBa_K739000 E X S X S P pSB1A2 pSB1A2

Bba_J23100

(constitutive promoter)

raxR (colR) raxH (colS)

BBa_K739000

mRFP

prax

BBa_K739000 E X S P

RICE GUARDIAN PROJECT: Exp. 2

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

• SDS-PAGE analysis of Ax21 on the membrane of E. Coli TOP10

<Lanes> M, Molecular weight standards; 1 and 2, E. coli transformed with pAT empty vector; 3 and 4, E. coli transformed with pAT-ax21 (no induction); 5 and 6, E. colitransformed with pAT-ax21 (arabinose induction); 1,3 and 5, soluble part; 2,4 and 6, insoluble part. 0.2% arabinose was used for induction.

RICE GUARDIAN PROJECT: Exp. 3

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

• Fluorescence and cell density of co-cultured media

* pAT: Cell-surface displaying vector * pAT-ax21: ax21 cloned in pAT vector * R.G.: Rice Guardian, engineered E. coli which expresses RaxH, RaxR, and mRFP .

Co-culture type Control pAT & R.G. pAT & R.G. pAT-ax21 & R.G. pAT-ax21 & R.G. Induction

• +
• +

RICE GUARDIAN PROJECT: Exp. 4

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

• Co-culture: Growth curve of each cell carrying different plasmid

• We proved that our parts successfully work !!!!!
• We plan to upgrade our current version to kill Xanthomonas oryzae.
• We expect our project may save a great number of starving people.

RICE GUARDIAN: Conclusion

2012 iGEM KOREA U SEOUL

PROJECT 1: RICE GUARDIAN

RICE GUARDIAN: a killer bacteria

BBa_J23100

(constitutive promoter)

Bacteriocin raxR (colR) raxH (colS) BBa_K739003

(prax)

BBa_K739000

PROJECT 2: BIOLOGICAL BINARY FULL ADDER

• What is a binary Adder?

a Digital Circuit that Performs Addition of Numbers

BINARY FULL ADDER PROJECT

2012 iGEM KOREA U SEOUL

PROJECT 2: LOGIC GATE

• Binary Full Adder – Basic Concept

BINARY FULL ADDER PROJECT

2012 iGEM KOREA U SEOUL

PROJECT 2: LOGIC GATE AND OR XOR

• XOR gate: from ICL iGEM 2008
• AND gate: from Davidson – Missouri Western 2007
• OR gate: modified XOR gate to create OR gate.

BINARY FULL ADDER PROJECT

• Final Construction of Bacterial Logic Gate

2012 iGEM KOREA U SEOUL

PROJECT 2: LOGIC GATE

BINARY FULL ADDER PROJECT

AND AND OR XOR XOR

BINARY FULL ADDER PROJECT : Characteristics

2012 iGEM KOREA U SEOUL

PROJECT 2: LOGIC GATE

• Differences between our full adder project and that of other teams

 Identical input, and output signal  System based on repeated simple structure  Unlimited combination of modules A A

• Final Construction of Bacterial Logic Gate

2012 iGEM KOREA U SEOUL

PROJECT 2: LOGIC GATE

BINARY FULL ADDER PROJECT

• Hill Equation: Express regulation of transcription
• Differential Equation : Express synthesis of proteins
• Solved Differential Equation

BINARY FULL ADDER PROJECT: FUNCTIONS

2012 iGEM KOREA U SEOUL

PROJECT 2: LOGIC GATE

BINARY FULL ADDER PROJECT: RESULT

• Concentration of Expressed Proteins

HUMAN PRACTICE

• BBa_K739000: ColRS
• BBa_K739003: promoter of raxRH + RBS + mRFP
• BBa_K739004: Ax21 protein - signaling part

2012 iGEM KOREA U SEOUL

HUMAN PRACTICE

OWNERSHIP: STANDARD PARTS

2012 iGEM KOREA U SEOUL

HUMAN PRACTICE

SHARING

Creative Challenger Program (CCP)

Described concept of synthetic biology and introduced Korea U Seoul’s iGEM activities.

Informing iGEM to Students

We introduced iGEM to junior highs who participated in Korea University biocamp.

Presentation Contest)

With a theme – Synthetic biology, new driving force for the future of Korea’s BT industry

2012 iGEM KOREA U SEOUL

HUMAN PRACTICE

EDUCATION

• Highschool Observer

2012 iGEM KOREA U SEOUL

HUMAN PRACTICE

REFERENCES

Sang-Wook Han, Pamela C. Ronald et al. 2011. Small protein-mediated quorum sensing in a gram- negative bacterium. PLoS ONE. 6:12

• A. Elings,,P.R. Reddy, T. Marimuthuc, W.A.H. Rossing, M.J.W. Jansene, P.S. Tengf, Ricebacterialleafblight:

field experiments, systems analysis and damage coefficients, Elsevier, volume 51, issue 2-1 Sang-Wook Han, Sang-Won Lee and Pamela C Ronald. 2011. Secretion, modification, and regulation of Ax21, Current Opinion in Microbiology. 14:62–67 Hyeok-Jin Ko, In-Geol Choi et al. 2012. Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes in Escherichia coli with a Novel Ligation Independent Cloning Vector Based on the Autotransporter YfaL. AEM. 10.1128 Baojun Wang, Martin Buck et al. 2011. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat Communication. 2:508 Nicolae RaduZabet et al. 2010. Proc. of the Alife XII Conference

ACKNOWLEDGEMENT

THANK YOU :^D

2012 iGEM KOREA U SEOUL