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Wits_CSIR iGEM 2011/ iGEM experience Presenter: Gloria Hlongwane PowerPoint PPT Presentation


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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 . Presentation Outline. Introduction to the iGEM competition iGEM Objectives

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Wits csir igem 2011 igem experience presenter gloria hlongwane

Wits_CSIRiGEM 2011/iGEM experience

Presenter: Gloria Hlongwane

School of Molecular and Cell Biology/

School of Chemical and Metallurgical Engineering ,

The University of the Witwatersrand, Johannesburg


Wits csir igem 2011 igem experience presenter gloria hlongwane

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


Wits csir igem 2011 igem experience presenter gloria hlongwane

INTRODUCTION TO THE iGEM COMPETITION

Elowitz,M.B., StanislasLeibler, 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

  • iGEM?

  • Started in 2003

  • Use of synthetic biology

  • BioBricks and the Registry of Standard Biological Parts


Wits csir igem 2011 igem experience presenter gloria hlongwane

iGEM Objectives

  • Make biology easy to engineer

  • Sharing of wisdom

  • Understand biological systems and Learn by building

  • Perform cutting edge research


Wits csir igem 2011 igem experience presenter gloria hlongwane

WITS_CSIR South Africa iGEM 2011 Team

Dr. Weinberg

Natasia

Ezio

Sasha

Michelle

Gloria


Wits csir igem 2011 igem experience presenter gloria hlongwane

iGEM 2011 Idea


Wits csir igem 2011 igem experience presenter gloria hlongwane

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


Wits csir igem 2011 igem experience presenter gloria hlongwane

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


Wits csir igem 2011 igem experience presenter gloria hlongwane

Aims

1. Standardise theophyllineriboswitch (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


Wits csir igem 2011 igem experience presenter gloria hlongwane

1. Standardise

Why?

How?

Fabricated thRS 1 & 2 via PCR

Standard biobrick prefix and suffix

  • 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


Wits csir igem 2011 igem experience presenter gloria hlongwane

2.Characterisation

Theophyllineriboswitch 1or 2

venus

[a]Fused both riboswitches to a Venus fluorescent protein

[b] Make testable constructs

Theophyllineriboswitch 1or 2

Double terminator B0015

venus

Strong promoter J23119

[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


Wits csir igem 2011 igem experience presenter gloria hlongwane

Results

8-fold

  • Promoter-thRS1-venus-double terminator

*

75

0

Promoter-RBS-CheZ-Venus-double terminator (K537013)

Promoter-thRS1-Venus-double terminator

Empty vector


Wits csir igem 2011 igem experience presenter gloria hlongwane

Results

  • Promoter-thRS2-venus-double terminator

2-fold

Promoter-RBS-cheZ-venus-double terminator (K537013)

Promoter-thRS2-Venus-double terminator

Empty vector

*

0

60


Wits csir igem 2011 igem experience presenter gloria hlongwane

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

Double terminator B0015

[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

Theophylline

riboswitch 1 or 2

Theophylline

riboswitch 1 or 2

cheZ N-fusion

venus C-fusion

cheZ N-fusion

venus C-fusion

Strong promoter J23119


Wits csir igem 2011 igem experience presenter gloria hlongwane

Results

  • Promoter-thRS1-cheZ-venus-double terminator

8-fold

Promoter-RBS-cheZ-venus-double terminator (K537013)

Promoter-thRS1-cheZ-venus-double terminator

*

Empty vector

0

105


Wits csir igem 2011 igem experience presenter gloria hlongwane

Results

  • Promoter-thRS2-cheZ-venus-double terminator

46-fold

*

Promoter-RBS-cheZ-venus-double terminator (K537013)

Promoter-thRS2-cheZ-venus-double terminator

Empty vector

0

135


Wits csir igem 2011 igem experience presenter gloria hlongwane

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.


Wits csir igem 2011 igem experience presenter gloria hlongwane

Achievements

  • A finalist at the European Jamboree : Won two prizes for Best poster and Best experimental approach.

  • Collaborated with ICL and were also invited to present in London at the Centre for Synthetic Biology and Innovation.

  • 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.


Wits csir igem 2011 igem experience presenter gloria hlongwane

Sponsors


Wits csir igem 2011 igem experience presenter gloria hlongwane

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, ChemBiol 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


Wits csir igem 2011 igem experience presenter gloria hlongwane

SUPPLEMENTARY INFORMATION


Wits csir igem 2011 igem experience presenter gloria hlongwane

SUPPLEMENTARY INFORMATION


Wits csir igem 2011 igem experience presenter gloria hlongwane

Chemotaxis

Che proteins facilitate conversion of ligand binding to the chemoreceptors into a change in flagella rotation.

  • In the presence of a chemical attractant, the bacterial movement is direct and characterised by longer runs and fewer tumbles.

  • Uniform environment, the two motions alternate in such a way that the cell moves in a random walk.

SUPPLEMENTARY INFORMATION


Wits csir igem 2011 igem experience presenter gloria hlongwane

TheophyllineRiboswitch (Topp and Gallivan, JACS, 2007)

  • Ligand-inducible CheZ expression system with an aptamer domain and an expression platform

  • 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 INFORMATION


Wits csir igem 2011 igem experience presenter gloria hlongwane

E

X

S

P

venus

  • Prefix=Restriction sites, EcoRI, NotI and XbaI

  • Suffix= SpeI, NotI and PstI

SUPPLEMENTARY INFORMATION


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