1. Wits_CSIR iGEM 2011/iGEM experience Presenter: Gloria Hlongwane School of Molecular and Cell Biology/ School of Chemical and Metallurgical Engineering, The University of the Witwatersrand, Johannesburg

2. Presentation Outline  Introduction to the iGEM competition  iGEM Objectives  WITS_CSIR South Africa iGEM 2011 Team  iGEM 2011 Idea  Research Background  Aims  Methods  Results  Concluding Remarks and Achievement

3. INTRODUCTION TO THE iGEM COMPETITION  iGEM?  Started in 2003  Use of synthetic biology  BioBricks and the Registry of Standard Biological Parts Elowitz,M.B., Stanislas Leibler, S.,Hacking DNA, (2009),giavasan.diludovico.it/.../HackingDNA, 7 April 2011 Medgadget, Synthetic biology (2006), medgadget.com/.../2006/11/igem_2006_winne.html, 7 April 2011 medgadget.com/.../2006/11/igem_2006_winne.html

4. iGEM Objectives  Make biology easy to engineer  Sharing of wisdom  Understand biological systems and Learn by building  Perform cutting edge research

5. WITS_CSIR South Africa iGEM 2011 Team Ezio Natasia Sasha Michelle Gloria Dr. Weinberg

6. iGEM 2011 Idea

7. Research Background  This study is an expansion of research on riboswitches (Gallivan’s lab; Emory, USA)  Gallivan and colleagues created theophylline- responsive riboswitch (Lynch et al., 2007)  Regulate cheZ, the global regulator of motility in E.coli (Topp and Gallivan, 2007 :cheZ knockouts restored the motility and were able to move up theophylline concentration gradient

8. Research Background  Topp and Gallivan for the 1st time showed that bacterial motility can be reprogrammed detect, trail and localise novel chemical signals  WITS_CSIR iGEM 2011 (BIOTWEET)  Lynch and Gallivan (2009) developed an improved riboswitch

9. Aims 1. Standardise theophylline riboswitch (thRS) 1 and 2 2. Characterise and quantify thRS1 and thRS2 3. Verify CheZ expression by thRS1 and establish if thRS2 can function as robust standardised “ON” switches that regulate cheZ

10. 1. Standardise Why?  Use by the WITS_CSIR 2011 iGEM team  Allow for easier future construction of cellular mimics that modulate bacterial chemotactic behaviour in response to theophylline How?  Fabricated thRS 1 & 2 via PCR  Standard biobrick prefix and suffix

11. 2.Characterisation Theophylline riboswitch 1or 2 venus Double terminator B0015 Strong promoter J23119 Theophylline riboswitch 1or 2 [a]Fused both riboswitches to a Venus fluorescent protein [b] Make testable constructs [c] Quantify the activation of these riboswitches when fused to the venus fluorescent reporter protein under variable theophylline concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM) over time using fluorometry

12. Results 8-fold * 75 0 Promoter-RBS-CheZ-Venus-double terminator (K537013) Promoter-thRS1-Venus-double terminator Empty vector  Promoter-thRS1-venus-double terminator

13. Results  Promoter-thRS2-venus-double terminator 2-fold * 060 Promoter-RBS-cheZ-venus-double terminator (K537013) Promoter-thRS2-Venus-double terminator Empty vector

14. 3. To establish if these theophylline sensitive riboswitches can function as robust standardised “ON” switches that regulate cheZ [a]Fused both riboswitches to CheZ-Venus fusion fluorescent protein [b] Make testable constructs [c] Quantify the activation of these riboswitches when fused to the cheZ- venus fluorescent fusion protein under variable theophylline concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM) over time using fluorometry venus C- fusion cheZ N-fusion Theophylline riboswitch 1 or 2 venus C-fusioncheZ N-fusion Theophylline riboswitch 1 or 2 Double terminator B0015 Strong promoter J23119

15. Results Promoter-RBS-cheZ-venus-double terminator (K537013) Promoter-thRS1-cheZ-venus-double terminator Empty vector  Promoter-thRS1-cheZ-venus- double terminator 0 105 8-fold *

16. Results 0 135 46-fold * Promoter-RBS-cheZ-venus-double terminator (K537013) Promoter-thRS2-cheZ-venus-double terminator Empty vector  Promoter-thRS2-cheZ-venus- double terminator

17. Concluding Remarks  thRS1 and thRS2 can regulate expression of the Venus protein thus inferring that functionality standardised construction units.  thRS1 and thRS2 can regulate expression of the CheZ- Venus fusion protein thus proving that both switches can function as robust standardised “ON” switches that regulate expression of CheZ.  thRS2 ˃ thRS1.

18. Achievements  Collaborated with ICL and were also invited to present in London at the Centre for Synthetic Biology and Innovation.  A finalist at the European Jamboree : Won two prizes for Best poster and Best experimental approach.  At the world championship in MIT acknowledged as one of the best top 16 projects to have been entered the iGEM competition worldwide in 2011.

19. Sponsors

20. References Lynch S.A. Desai S.K.,Sajja,H.K., and Gallivan J.P.A high-trhoughput screen for synthetic riboswitches reveals mechanistic insight into their function. 2007, Chem Biol 14:173-184 Topp S, Gallivan JP. Guiding bacteria with small molecules and RNA. J Am Chem Soc 2007;129:6807- 11 Topp S. and Gallivan J.P. Random walks to synthetic riboswitches – a high throughput selection based on cell motility. 2008, ChemBiochem 9:210-213

21. SUPPLEMENTARY INFORMATION

23.  In the presence of a chemical attractant, the bacterial movement is direct and characterised by longer runs and fewer tumbles. Che proteins facilitate conversion of ligand binding to the chemoreceptors into a change in flagella rotation. Chemotaxis  Uniform environment, the two motions alternate in such a way that the cell moves in a random walk.

24. SUPPLEMENTARY INFORMATION  Ligand-inducible CheZ expression system with an aptamer domain and an expression platform Theophylline Riboswitch (Topp and Gallivan, JACS, 2007)  Binding of the ligand to aptamer leads to a conformational change in the secondary structure of the RNA THUS exposing the RBS followed by the activation of the target gene

25. SUPPLEMENTARY INFORMATION  Prefix=Restriction sites, EcoRI, NotI and XbaI  Suffix= SpeI, NotI and PstI venus EX S P