Team: IIT Madras 440000 new cases of Multiple Drug Resistant - - PowerPoint PPT Presentation

Team: IIT Madras

440000 new cases of Multiple Drug Resistant Tuberculosis

 Overuse of Antibiotics

• Cattle/Poultry Feed
• Prescriptions

 Antibiotic resistance genes in research

RIP RIP

Selection against non‐ transformants

• Antibiotic Resistance

Genes

• Risk of Horizontal

Gene Transfer

Selects for transformants

• Metabolic Selection

Markers

Project Sunscreen

P R P R P R H+ H+ H+ H+ H+ + ATP Retinal Retinal ATP Synthase

Project Artemis

Electron Transport Chain H+ H+ H+ H+ Glycolysis Carbon Source ATP ATP Synthase

Electron Transport Chain H+ P R P R P R H+ H+ H+ H+ H+ Glycolysis + Carbon Source ATP Retinal Retinal ATP Synthase

Recombinant Protein

BBa_K572005 Proteorhodopsin Coding Gene BBa_K572005 BBa_B0034 BBa_B0015 BBa_K572006 Proteorhodopsin Generator BBa_K572005 BBa_B0034 BBa_B0015 T7 Promoter BBa_K572007 IPTG induced Proteorhodopsin Generator (pET vector) BBa_K572005 BBa_B0034 BBa_J23119 BBa_B0015 BBa_K572008 Constitutive Proteorhodopsin Generator

Range of Tm, %GC and Length

INPUTS

OUTPUTS Number and positions of Mutations Protein sequence Primer sequence Tm and GC content Common primer for mutation sites closer than 20bp

PRIMER TOOL PRIMER TOOL

BBa_K572009 Mouse Beta‐Carotene Dioxygenase Coding Gene BBa_K572009 BBa_B0034 BBa_B0015 Dioxygenase Generator BBa_K572009 BBa_B0034 BBa_J23119 BBa_B0015

CrtEBIY

Retinal Biosynthesis

β‐Diox

BBa_K274210: Endogenous synthesis of beta‐carotene BBa_K572009: Mouse Beta Carotene Dioxygenase converts beta carotene to Trans‐Retinal Retinal with PR in presence

• f light helps in development
• f proton gradient

0.2 0.4 0.6 0.8 1

IPTG+ Light‐ IPTG+ Light+

Relative number of CFUs Azide Negative Samples Azide Treated Samples

K572005 Over Expression of GPR in DH5α

.

15% SDS PAGE gel showing the IPTG induction of PR expression

Time after induction

28kDA K572005 Functional Studies ‐ AzideTest

.

P S X E

BBa_K572100

Proteorhodopsin

BBa_K572005

Backbone of pSB1C3, without Chloramphenicol Resistance Gene

*Adam M Feist et al 2007, A genome‐scale metabolic reconstruction for Escherichia coli K‐12 MG1655 that accounts for 1260 ORFs and thermodynamic information, Molecular Systems Biology ** Walter et al 2006, “Light empowering Escherichia coli with proteorhodpsin”, PNAS

SBML*

• Quantification of Proton flux through proteorhodopsin
• E.coli genome scale model extended to include proteorhodopsin

COBRA

• Constraint based Reconstruction and Analysis
• Objective function optimization (Flux)

Model

• Simulations to validate network using experimental data

Analysis

• Comparative analysis of increase in growth rate in proteorhodopsin transformed

cells

Hypothesis: Increase in growth rate due to Proteorhodopsin proton efflux in minimal carbon media



Simulation of published experimental data for model validation



Jessica M. Walter et al 2006, Light‐powering Escherichia coli with proteorhodopsin, PNAS) “Light‐driven pumping by PR can fully replace respiration as a cellular energy source in some environmental conditions”

20 40 60 80 100 120 5 10 15

% Increase in growth rate Glucose uptake flux (in mmol/ g‐dw hr)

Complete Inhibition of ETC in presence of azide

5 10 15 20 25 30 5 10 15

% Increase In growth rate Glucose uptake flux (in mmol/g‐dw hr)

70% Inhibition of ETC in presence of azide

5 10 15 20 25 30 35 5 10 15

% Increase in growth rate Glucose uptake flux ( in mmol/g‐dw hr)



Comparative analysis of increase in growth rate in proteorhodopsin transformed cells

Growth rate advantage due to PR

0.5 1 1.5 2 2.5 2 4 6 8 10 Cell concentration (g/l) Time (Hour)

Control Growth Profiles (DH5α)

LB 0.2 g/l 0.4 g/l 1 g/l 3 g/l 5 g/l 0.5 1 1.5 2 2.5 3 2 4 6 8 Specific growth rate (Hour ‐1) time(hour)

Comparison of rate of change of specific growth rate in LB

IPTG Induced PR+pET41a in LB PR+pET41a Control in LB y = 9.1994e‐0.711x R² = 0.9836 y = 4.2768e‐0.551x R² = 0.9881 1 2 3 2 4 6 8 Specific Growth rate (Hour‐1) Time (Hour)

Comparison of specific growth rate in M9 media

IPTG Induced5g/l M9 media 5g/l M9 media Control

BBa_K081014/mRFP1 gen BBa_K118011 (PcstA) Carbon Stress Induced Promoter

BBa_K572001 : Carbon Stress Response Indicator

Substrate Availability

Time Bba_K572001

525 nm 584 nm

Wavelength

RFP GPR

Absorption Spectrum

100 200 300 400 500 600 700 2 4 6 8 RLU/OD600 Time (Hours)

RFP Expression

0.02 0.1 0.5 LB LB+0.2

As viewed under a fluorescence microscope, RFP being expressed in DH5α with PcstA promoter

1 2 3 4 5 6 7 8 9 10 2 4 6 8 10 OD600 Time (Hours)

Growth Profile

0.02 0.1 0.5 LB LB+0.2

P S X E

BBa_K572200

Proteorhodopsin

BBa_K572005

Backbone of pSB1C3, without Chloramphenicol Resistance Gene

pSB1Pe – Expression Vector

PcstA

 Kwang‐Hwan "Kevin" Jung, Ph.D., Associate Professor,

Department of Life Science and Institute of Biological Interfaces, Sogang University, Korea for sending us the plasmid (pKJ900) with proteorhodopsin gene.

 Prof. Karthik Raman of IIT Madras who helped our team

in efficient modelling of our Project

 Mr. Shrikumar Suryanarayan, Adjunct Professor, IITM, for

his financial assistance and encouragement

 Prof. K B Ramachandran and Prof. Mukesh Doble,

Department of Biotechnology IIT Madras for their constant support and encouragement

 IITM and IITMAA(IITM Alumini Association) for their

financial support