“Super Silencer” iGEM 2011 HKU-Hong Kong Team Outline Inspiration Our Aim The Project Design of the Project Structural Basis of the Super Silencer Experimental Approach Challenges faced and Strategy of Plan Results Summary Biobricks Human Practices Inspiration Virulent genes Inspiration • Gene silencing? RNAi in eukaryotes • Prokaryotes? Gene deletion Irreversible Essential genes: lethal Vision • Gene silencing in prokaryotes! Reversible Multiple targeting Ideas • Spreading effect of heterochromatin • H-NS Histone-like nucleoid structuring protein (H-NS) • Found in nucleoid • DNA binding • Gene repression HNS Structural basis • H-NS: naturally-occurring in Gram –ve bacteria • N-terminal domain (a.a. 1-64): capable of dimerisation • Flexible Linker domain (a.a. 65- 89) • C-terminal domain (a.a. 90-137): binding to curved DNA H-NS (Full length,137a.a.)dimer Our Aims • Utilize H-NS repression to achieve gene repression • Incorporate specificity by a DNA binding domains, tetR. Combine H-NS and tetR, i.e. Fusion protein of tetR::H-NS tetR Structural basis • tetR: tetracycline repressor protein • 10 -helices • 5 - 10: tetracycline(inducer) binding domain • 1 - 3: DNA binding domain tetR dimer binding on tetO site tetR::HNS Structural basis • Fusion protein construct: tetR::H-NS • tetR bind to tetO • H-NS domain oligomerises with wild type H-NS tet::H-NS fusion protein binding with tetO site Project overview • Gene silencing in prokaryotes • Fusion protein construct: tetR::HNS • Principle testing: repression of GFP The Experiments • Part 1 :Making fusion protein of tetR::HNS and related parts J23XXX-R/RH/RHH ▪ R=tetR, RH=tetR::HNS, RHH= tetR::HNS-RBS-HNS ▪ J23XXX: Constitutive promoter family The Experiments • Part 2: Incorporate the recognition site of tetR--- tetO • PCR tetO sites into different place among the plasmid-promoter-GFP • Attenuated the plasmid-promoter-tetO-GFP into MG1655 genome Insertion of the tetO2-GFP sites into the E.Coli MG1655 genome (attTn7 site) Challenges we faced 1.Ribosome binding sites Finally decided to use BBa_B0034 in our project as its strength is one of the highest in the Registry collection. 2. Gel shift assay Used to detect the presence of DNA-Protein interaction between our fusion protein and the recognition sites. An important follow-up step Strategy of the Plan • When using plasmid pEGFP-tetO2-X to test for repression: no satisfactory result 1. tetR unfit plasmid DNA structure 2. Plasmid lost from E.coli Recombination of tetO2-0-EGFP, tetO2-1-EGFP, tetO2- 0-sfGFP in to E.Coli MG1655 genome (attTn7 site) Results * J23103, J23109, J23116, J23109 are constitutive promoter family Results Confirmation data of promoter strength Results Summary Biobricks 1. Tetracycline repressor selection --- (Constitutive Promoter Family + RBS + tetR ) K544001 – (Promoter: J23103) K544002* - (Promoter: J23109) K544003* - (Promoter: J23116) K544012 - (Promoter: J23106) 2. Super silencer selection --- (Constitutive Promoter Family + RBS + tetR-HNS(2-90) ) K544011 - (Promoter: J23103) K544013* - (Promoter: J23109) K544014 - (Promoter: J23106) Dimeric Keima-Red Fluorescent Protein --- (dKeima) 3. Reporter K544021 - Dimeric version of the fluorescent protein “Keima-Red,” as an alternative reporter Further Work • Gel mobility assay to detect tetR::HNS interaction with DNA • Adding the inducer chlortetracycline(CTC) to determine whether the repression can be relieved ▪ CTC: induce conformational change of tetR tetR release from tetO • Devise better ways to integrate the construct into the bacterial genome Prospects • Novel tool of gene silencing in prokaryotes • Silence virulent genes Human Practices • Aim Introduce synthetic biology Introduce iGEM and our team Provide hands-on lab experience Raise participants’ interest in the biosciences Human Practices • Open Day • Secondary school workshop Acknowledgements We would like to express our heartfelt gratitude to • Our instructors Dr. J.D. Huang, Dr. Huang Wei, and Dr. Xuechen Li • Postgraduates Mr. Li Sihong, Mr. Liu lizhong, and Mr. Zhang Baozhong • Our sponsors HKU Faculty of Science, HKU Department of Biochemistry, AstraZeneca HK Ltd, HKU School of Biological Sciences, Bio-Rad, Life technologies • Our fellow team memebers Q & A References • Rimsky S. Structure of the histone-like protein H-NS and its role in regulation and genome superstructure. Current Opinion in Microbiology.2004 Apr;7(2):109-14. • Dorman CJ. H-NS: A universal regulator for a dynamic genome. Nature Reviews Microbiology .2004 May;2(5):391-400. • Dame RT, Wyman C, Wurm R, Wagner R, Goosen N. Structural Basis for H- NS-mediated Trapping of RNA Polymerase inthe Open Initiation Complex at the rrnBP1. The Journal of biological chemistry.2002 Jan 18;277(3):2146-50. • Peter Orth, Dirk Schnappinger, Wolfgang Hillen, Wolfram Saenger & Winfried Hinrichs.Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature Structural Biology 7, 215 - 219 (2000) • Kevin F. Murphy, Gábor Balázsi, James J. Collins. Combinatorial promoter design for engineering noisy gene expression. Proceedings of the National Academy of Sciences of the United States of America. Volume: 104, Issue: 31, Pages: 12726-12731 (2007) .