iGEM 2015 Mycobacterium Revelio TEAM IISER PUNE
From Muggles to Wizards
Muggle Studies Jehangir Hospital, Pune ZN- staining LJ media culturing MGIT technique Image: www.scilogs.com http://medind.nic.in www.tbonline.info
Muggle Studies Cepheid GeneXpert GenePath Dx lab
Our Goal Detection of Mycobacterium using a genetic device : • Quick • Robust • Economic • Safe
The three magical spells Gemino The Doubling Charm Aparecium The Visibility Charm http://animationsa2z.com/harry-potter.php Avada Kedavra The Killing Curse
Gemino The Doubling Charm Overclocking the cell cycle
Gemino in Harry Potter Video from Harry Potter movie showing Gemino curse
The Gemino Analogy Target • Cell cycle controlling proteins • Oscillating concentrations during Behavior cell cycle period Gemino • Enhance the frequency of these oscillations • More number of cell cycles Resultant activated during same time period Genetic Gemino Oscillator
The Fast, Robust,Tunable Oscillator Stricker et al. (2008) +arabinose AraC GFP LacI -IPTG Hybrid Promoter – p lac/ara1
Oscillator Construct ready!
Variable Link Oscillator Hasty et al (Chaos 2002) P RM P RM c1 GFP C1 Protein RCSA P RM rcsA Protease
Original Model Two Plasmid System Our simulation to check the range of (m x : m y ) values for which oscillations occur - y x m x > m y Equations from literature : dx dt = m x f ( x ) - g x x - g xy xy dy = m y f ( x ) - g y y dt dg dt = m x f ( x ) - g g g - g gy gy Production terms Degradation terms The protease effect!
Single Plasmid System Our Model Bio-brick part (RBS) Efficiency BBa_B0034 1.0 (Reference) x y BBa_B0030 0.6 BBa_B0031 0.07 m ' x = m ' y = m ' e x : e y = 0.6 : 0.07 = 8.75:1 e x > e y e x : e y :: m x : m y dx dt = e x ( m ' f ( x )) - g x x - g xy xy dy = e y ( m ' f ( x )) - g y y dt dg dt = e x ( m ' f ( x )) - g g g - g gy gy RBS Efficiency Factors
The three magical spells Gemino The Doubling Charm Aparecium The Visibility Charm http://animationsa2z.com/harry-potter.php Avada Kedavra The Killing Curse
How do we see them?
Aparecium The Visibility Charm Making the invisible visible
Aparecium in Harry Potter
The Aparecium Analogy Invisible Cells Visible Cells β -carotene Lycopene
Detection Techniques Microscopic Macroscopic http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-imager-for-polarized-light.html http://www.thermoscientific.com/en/product/varioskan-flash-multimode-reader.html
Measurement and Characterization of Pigments Using Spectroscopy Carotene Lycopene Optical methods for noninvasive determination of carotenoids in human and animal skin - Maxim E. Darvin (2013)
Measurement and Characterization of Pigments Using Microscopy Beta carotene biobrick in E.coli MG1655 cells Control E.coli MG1655 cells Lycopene biobrick in E.coli MG1655 cells Beta carotene biobrick in E.coli TOP10 cells Lycopene biobrick in E.coli TOP10 cells
Further Characterization Plans Further in Microscopy : • Use of colour filters in microscope • Use of portable and cost efficient microscope like • Foldscope • Paperscope http://www.zeiss.com/microscopy/en_de/service-support/filtersets-dyes/_jcr_content/stagepar/stage/slide/stageimage/image.img.jpg/1364973094776.jpg/filter-sets-stage.jpg
Further Characterization Plans Testing Foldscope with slide containing blood smear stained with giemsa dye for it’s magnification power High Magnification Low Magnification beta tester lab
Again to the Muggles
The three magical spells Gemino The Doubling Charm Aparecium The Visibility Charm http://animationsa2z.com/harry-potter.php Avada Kedavra The Killing Curse
Now we need to stop them!
Avada Kedavra The Killing Curse
Avada Kedavra in Harry Potter Video from Harry Potter movie showing Avada Kedavra curse
The Avada Kedavra Analogy AHL LuxR I LuxR LuxR LuxI p(tetR) LacI+pL CcdB CcdB Lux pR
The Test Constructs Receiver Sender Bba_R0011+Bba_K805016 Bba_T9002 – pSB1C3 - pSB1K3
And the circuit works! All the cultures are induced with IPTG Scale bar- 5um
Measurement and Characterisation
When is the terminator triggered?
At threshold cell density, terminator is switched on Termination for switch based mechanism Normal Termination Circuit
The three magical spells Gemino The Doubling Charm Aparecium The Visibility Charm http://animationsa2z.com/harry-potter.php Avada Kedavra The Killing Curse
Our Device Phage containing genetic device attack M.tuberculosis Genetic device enters the cell and takes control of the Genetic device cell cycle added to sample Rapid division and pigment production Pigment detected Termination “Self Destruct”
Our Prototype Cap Temporary Seal Protective foil Sample Mycobacteriophage One way screw cap Breaking foil Mixing sample with phage
Achievements Avada Aparecium Gemino • Pigmented cells Kedavra • Modelling shows obtained oscillations! • Assembled • Sender device in Characterized the • 3 plasmids pSB1K3 biobricks by containing various backbone Cambridge Team parts of the oscillator module • • Showed that Assembled ready Sender and foldscope to Receiver devices overcome the • Submitted 2 work in co- issue of expensive biobricks central culture! microscopy to the oscillator design • Characterized the working of these Biobricks by analyzing GFP output
Team PI : Dr. Chaitanya Athale (1 st from left) Mentor : Neha Khetan (3 rd from left) Manasi Gangan (4 th from left)
Acknowledgements Attributions - Dr. Vikram Padbidri, Jehangir Hospital - Dr. Mughda Gadgil, NCL - Dr. Kavita Khatod, GenePath Dx Lab - Dr. Nishad Matange, IISER Pune - Dr. Gayathri Pananghat, IISER Pune
Thank You
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