Wits_CSIR iGEM 2011/ iGEM experience Presenter: Gloria Hlongwane

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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

1Presentation 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

2INTRODUCTION TO THE iGEM COMPETITIONiGEM?

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 20113iGEM ObjectivesMake biology easy to engineer

Sharing of wisdom

Understand biological systems and Learn by building

Perform cutting edge research 4WITS_CSIR South Africa iGEM 2011 Team

EzioNatasiaSashaMichelleGloriaDr. Weinberg5

iGEM 2011 Idea6Research Background This study is an expansion of research on riboswitches (Gallivans 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

7Research Background Topp and Gallivan for the 1st time showed that bacterial motility can be reprogrammed

detect, trail and localise novel chemical signalsWITS_CSIR iGEM 2011 (BIOTWEET)

Lynch and Gallivan (2009) developed an improved riboswitch8Aims 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 cheZ91. 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 theophyllineHow? Fabricated thRS 1 & 2 via PCR

Standard biobrick prefix and suffix102.Characterisation Theophylline riboswitch 1or 2venus venus Double terminator B0015 Strong promoter J23119Theophylline 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

11 Results

8-fold*750Promoter-RBS-CheZ-Venus-double terminator (K537013)Promoter-thRS1-Venus-double terminator Empty vectorPromoter-thRS1-venus-double terminator12 Results Promoter-thRS2-venus-double terminator

2-fold*060Promoter-RBS-cheZ-venus-double terminator (K537013)Promoter-thRS2-Venus-double terminator Empty vector133. 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-fusionTheophylline riboswitch 1 or 2venus C-fusion cheZ N-fusionTheophylline riboswitch 1 or 2Double terminator B0015 Strong promoter J2311914 Results Promoter-RBS-cheZ-venus-double terminator (K537013)Promoter-thRS1-cheZ-venus-double terminator Empty vectorPromoter-thRS1-cheZ-venus-double terminator

01058-fold*15 Results

013546-fold*Promoter-RBS-cheZ-venus-double terminator (K537013)Promoter-thRS2-cheZ-venus-double terminator Empty vectorPromoter-thRS2-cheZ-venus-double terminator16 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.

17AchievementsCollaborated 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.

18 Sponsors

19 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 14:173-184Topp S, Gallivan JP. Guiding bacteria with small molecules and RNA. J Am Chem Soc 2007;129:6807-11Topp S. and Gallivan J.P. Random walks to synthetic riboswitches a high throughput selection based on cell motility. 2008, ChemBiochem 9:210-213

20SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION

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. ChemotaxisUniform environment, the two motions alternate in such a way that the cell moves in a random walk.SUPPLEMENTARY INFORMATIONLigand-inducible CheZ expression system with an aptamer domain and an expression platformTheophylline 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

SUPPLEMENTARY INFORMATIONPrefix=Restriction sites, EcoRI, NotI and XbaI

Suffix= SpeI, NotI and PstI venusEXSP