“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)