Mycobacterium Revelio TEAM IISER PUNE From Muggles to Wizards - - PowerPoint PPT Presentation

iGEM 2015

Mycobacterium Revelio

TEAM IISER PUNE

From Muggles to Wizards

Jehangir Hospital, Pune

Image: www.scilogs.com http://medind.nic.in www.tbonline.info

ZN- staining LJ media culturing MGIT technique

Muggle Studies

GenePath Dx lab

Muggle Studies

Cepheid GeneXpert

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

Avada Kedavra

The Killing Curse

http://animationsa2z.com/harry-potter.php

Overclocking the cell cycle

Gemino

The Doubling Charm

Gemino in Harry Potter

Video from Harry Potter movie showing Gemino curse

The Gemino Analogy

• Cell cycle controlling proteins

Target

• Oscillating concentrations during

cell cycle period

Behavior

• Enhance the frequency of these
• scillations

Gemino

• More number of cell cycles

activated during same time period

Resultant

Gemino

Genetic Oscillator

The Fast, Robust,Tunable Oscillator

AraC GFP LacI

+arabinose

• IPTG

Stricker et al. (2008)

Hybrid Promoter – plac/ara1

Oscillator Construct ready!

Variable Link Oscillator

Hasty et al (Chaos 2002)

c1 GFP rcsA PRM PRM PRM

C1 Protein RCSA Protease

Original Model

dx dt = mx f (x)-g xx -g xyxy dy dt = my f (x)-g yy dg dt = mx f (x)-g gg-g gygy

Equations from literature :

x

y

Two Plasmid System

mx > my

Production terms Degradation terms The protease effect!

Our simulation to check the range of (mx: my) values for which oscillations occur -

x y

dx dt = ex(m' f (x))-g xx -g xyxy dy dt = ey(m' f (x))-g yy dg dt = ex(m' f (x))-ggg-ggygy m'x = m'y = m' ex > ey ex :ey :: mx : my

Single Plasmid System Our Model RBS Efficiency Factors

Bio-brick part (RBS) Efficiency BBa_B0034 1.0 (Reference) BBa_B0030 0.6 BBa_B0031 0.07

ex : ey = 0.6 : 0.07 = 8.75:1

The three magical spells

Gemino

The Doubling Charm

Aparecium

The Visibility Charm

Avada Kedavra

The Killing Curse

http://animationsa2z.com/harry-potter.php

How do we see them?

Making the invisible visible

Aparecium

The Visibility Charm

Aparecium in Harry Potter

β-carotene Lycopene

The Aparecium Analogy

Invisible Cells Visible Cells

Detection Techniques

Macroscopic

http://www.thermoscientific.com/en/product/varioskan-flash-multimode-reader.html http://www.zeiss.com/microscopy/en_de/products/light-microscopes/axio-imager-for-polarized-light.html

Microscopic

Measurement and Characterization

• f Pigments

Carotene Lycopene

Using Spectroscopy

Optical methods for noninvasive determination of carotenoids in human and animal skin - Maxim E. Darvin (2013)

Measurement and Characterization of Pigments

Using Microscopy

Control E.coli MG1655 cells Lycopene biobrick in E.coli TOP10 cells Beta carotene biobrick in E.coli TOP10 cells Lycopene biobrick in E.coli MG1655 cells Beta carotene biobrick in E.coli MG1655 cells

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

Further in Microscopy :

• Use of colour filters in microscope
• Use of portable and cost efficient microscope like
• Foldscope
• Paperscope

Testing Foldscope with slide containing blood smear stained with giemsa dye for it’s magnification power

beta tester lab

Low Magnification High Magnification

Further Characterization Plans

Again to the Muggles

The three magical spells

Gemino

The Doubling Charm

Aparecium

The Visibility Charm

Avada Kedavra

The Killing Curse

http://animationsa2z.com/harry-potter.php

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

LuxR LuxI

LuxR

I

LuxR

CcdB

Lux pR

CcdB AHL LacI+pL p(tetR)

The Avada Kedavra Analogy

The Test Constructs

Sender Receiver

Bba_R0011+Bba_K805016

• pSB1K3

Bba_T9002 – pSB1C3

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

Avada Kedavra

The Killing Curse

http://animationsa2z.com/harry-potter.php

Termination “Self Destruct” Rapid division and pigment production Genetic device enters the cell and takes control of the cell cycle Phage containing genetic device attack M.tuberculosis Genetic device added to sample Pigment detected

Our Device

Our Prototype

Temporary Seal Protective foil Mycobacteriophage Sample Cap One way screw cap Breaking foil Mixing sample with phage

Achievements

Gemino

• Modelling shows
• scillations!
• 3 plasmids

containing various parts of the

• scillator module

ready

• Submitted 2

biobricks central to the oscillator design

Aparecium

• Pigmented cells
• btained
• Characterized the

biobricks by Cambridge Team

• Assembled

foldscope to

• vercome the

issue of expensive microscopy

Avada Kedavra

• Assembled

Sender device in pSB1K3 backbone

• Showed that

Sender and Receiver devices work in co- culture!

• Characterized the

working of these Biobricks by analyzing GFP

• utput

Team

PI : Dr. Chaitanya Athale (1st from left) Mentor : Neha Khetan (3rd from left) Manasi Gangan (4th 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