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 Bacterial leaf blight (BLB) • >90% of total rice produced • Caused by Xanthomonas in Asia • One of the most serious • Most important crop for disease of rice human – providing 30~85 % of total calories consumed by Asian people – 8% of food energy for 1 billion people over the world (except Asia) http://eprints.hec.gov.pk/2349/1/2204.htm http://books.irri.org / 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
Xanthomonas oryzae and Ax21 Ax21 Bacterial Leaf Blight Xanthomonas oryzae Rice Sang-Wook Han, Pamela C. Ronald et al. 2011. Small protein-mediated quorum sensing in a gram-negative bacterium. PLoS ONE. 6:12 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
Xanthomonas oryzae and Ax21 Ax21 CLEAR Xanthomonas Xanthomonas oryzae RICE GUARDIAN Sang-Wook Han, Pamela C. Ronald et al. 2011. Small protein-mediated quorum sensing in a gram-negative bacterium. PLoS ONE. 6:12 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
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 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
RICE GUARDIAN: Part Design Ax21 Sensing bacteria BBa_K739000 BBa_J23100 BBa_K739003 (constitutive raxR raxH (p rax ) promoter) mRFP (colR) (colS) RaxR/RaxH : a two component regulatory system in Xanthomonas which works as a receptor for Ax21. 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
RICE GUARDIAN: Part Design Ax21 Sensing bacteria Ax21 RaxH BBa_J23100 BBa_K739003 (constitutive (p rax ) raxR raxH RaxR promoter) mRFP (colR) (colS) 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
RICE GUARDIAN: Part Design • Problem - Xanthomonas is pathogenic solution: Overexpressing Ax21 inside E. Coli Problem: We could not attain Ax21 in a soluble form solution: DISPLAYING Ax21 on cell membrane pBAD BBa_K739004 (ax-21) Ax21 * T domain : Translocator domain Displaying T domain Bacteria pAT: a vector designed to display heterologous proteins on cell-surface of E. coli 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 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
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 • Cloning: PCR from gDNA + 3A assembly Bba_J23100 BBa_K739000 BBa_K739000 (constitutive raxR raxH promoter) p rax (colR) (colS) mRFP E X S X S P pSB1A2 Bba_J23100 BBa_K739000 BBa_K739000 (constitutive raxR raxH promoter) p rax (colR) (colS) mRFP E X S P pSB1A2 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
RICE GUARDIAN PROJECT: Exp. 2 • SDS-PAGE analysis of Ax21 on the membrane of E. Coli TOP10 <Lanes> 1,3 and 5, soluble part; M, Molecular weight standards; 2,4 and 6, insoluble part. 1 and 2, E. coli transformed with pAT empty vector; 0.2% arabinose was used for 3 and 4, E. coli transformed with pAT-ax21 (no induction); induction. 5 and 6, E. coli transformed with pAT-ax21 (arabinose induction); 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
RICE GUARDIAN PROJECT: Exp. 3 • 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 Control pAT pAT pAT-ax21 pAT-ax21 type & R.G. & R.G. & R.G. & R.G. Induction - - + - + 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
RICE GUARDIAN PROJECT: Exp. 4 • Co-culture: Growth curve of each cell carrying different plasmid 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
RICE GUARDIAN: Conclusion • We proved that our parts successfully work !!!!! • We plan to upgrade our current version to kill Xanthomonas oryzae. BBa_K739000 BBa_J23100 RICE BBa_K739003 (constitutive raxR raxH (p rax ) GUARDIAN: promoter) Bacteriocin (colR) (colS) a killer bacteria • We expect our project may save a great number of starving people. 2012 iGEM KOREA U SEOUL PROJECT 1: RICE GUARDIAN
PROJECT 2: BIOLOGICAL BINARY FULL ADDER
BINARY FULL ADDER PROJECT • What is a binary Adder? a Digital Circuit that Performs Addition of Numbers 2012 iGEM KOREA U SEOUL PROJECT 2: LOGIC GATE
BINARY FULL ADDER PROJECT • Binary Full Adder – Basic Concept XOR OR AND 2012 iGEM KOREA U SEOUL PROJECT 2: LOGIC GATE
BINARY FULL ADDER PROJECT • 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 XOR XOR AND OR AND 2012 iGEM KOREA U SEOUL PROJECT 2: LOGIC GATE
BINARY FULL ADDER PROJECT : Characteristics • 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 2012 iGEM KOREA U SEOUL PROJECT 2: LOGIC GATE
BINARY FULL ADDER PROJECT • Final Construction of Bacterial Logic Gate 2012 iGEM KOREA U SEOUL PROJECT 2: LOGIC GATE
BINARY FULL ADDER PROJECT: FUNCTIONS • Hill Equation: Express regulation of transcription • Differential Equation : Express synthesis of proteins • Solved Differential Equation
BINARY FULL ADDER PROJECT: RESULT • Concentration of Expressed Proteins 2012 iGEM KOREA U SEOUL PROJECT 2: LOGIC GATE
HUMAN PRACTICE
OWNERSHIP: STANDARD PARTS • BBa_K739000: ColRS • BBa_K739003: promoter of raxRH + RBS + mRFP • BBa_K739004: Ax21 protein - signaling part 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 2012 iGEM KOREA U SEOUL HUMAN PRACTICE
ACKNOWLEDGEMENT
THANK YOU :^D 2012 iGEM KOREA U SEOUL
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