and E nhanced e X pression Team: IIT Madras Indian Institute of - - PowerPoint PPT Presentation

Photonivorous Bacteria for Resolution and Enhanced eXpression

Team: IIT Madras Indian Institute of Technology Madras

Selection Markers for Screening

Antibiotic Resistance

Negative selection markers More risky horizontal gene transfer

Metabolic Markers

Positive selection markers Limited strains of cells that can be used

Proteorhodopsin

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

Oxidative Phosphorylation

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

Electron Transport Chain H+ Glycolysis Carbon Source ATP Synthase

Photophosphorylation to the rescue

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

Minimal Media

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

Minimal Media with PR

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

Recombinant protein production in Minimal Media

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

Recombinant Protein

Recombinant protein production in Minimal Media

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

Recombinant Protein

SunScreen – Light based Screening of Clones

Minimal Media

(containing Retinal)

Project Sunscreen - Design

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 BBa_K572005 BBa_B0034 BBa_J23119 BBa_B0015 BBa_K572008 Constitutive Proteorhodopsin Generator

INPUTS

• Protein sequence
• Number and positions
• f Mutations
• Range of Tm, %GC and

Length of primers

OUTPUTS

• Primer Sequence
• Tm, %GC and

Length PRIMER

Characterization of Proteorhodopsin

0.2 0.4 0.6 0.8 1

IPTG+ Light- IPTG+ Light+

Relative number of CFUs Azide Negative Samples Azide Treated Samples

Azide Test on BL21 with PR

K572005 Over Expression of GPR in BL21DE3

.

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

Time after induction

28 kDA

Project Sunscreen - Design

BBa_K572009 Mouse Beta-Carotene Dioxygenase Coding Gene BBa_K572009 BBa_B0034 BBa_B0015 Dioxygenase Generator BBa_K572009 BBa_B0034 BBa_J23119 BBa_B0015 Constitutive Dioxygenase generator

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

pSB1Pc – Cloning Vector

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”

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

Proteorhodopsin

ATP Synthase Genomic Scale Metabolic Pathway

H+ H+ H+ H+

Model - Design

*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”

SBML

• Network Composition (*, **)
• Reaction and Specie parameters

COBRA

• SBML toolbox, MATLAB
• Setting up model in COBRA (readCbModel)

Model • Model validated using literature data Analysis • Growth rate variation from WT to PR

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

Metabolic Modeling

Glucose Uptake flux (mmol/g-dw hr) Growth Rate

(hr-1)

Model_WT Growth Rate

(hr-1)

Model_PR Complete Inhibition (Azide)

2 3 0.0035 4 0.0257 0.0306 5 0.0528 0.0577 6 0.0799 0.0848 8 0.134 0.1389 10 0.1882 0.1931 12 0.2423 0.2472

70% Inhibition (Azide)

2 0.1576 0.16 3 0.2449 0.2473 4 0.3322 0.3346 5 0.4195 0.422 6 0.5068 0.5093 8 0.6785 0.6831 10 0.7609 0.7655 12 0.8433 0.8479

Model

Simulations for Validations

Model

Simulations to find minimal media

Glucose uptake Rate (mmol / g-dw hr) model_WT model_PR 0.35 0.0014 0.4 0.0042 0.0062 0.5 0.0139 0.016 0.6 0.0236 0.0257 0.9 0.0528 0.0548 2 0.1589 0.1609 3 0.2552 0.2572 4 0.3515 0.3535 5 0.4478 0.4499 6 0.5441 0.5461 8 0.7367 0.7387 10 0.8856 0.8901 12 0.968 0.9725

Effectors of Growth Rate

10 20 30 40 50

Global Negative (R-I-A-L-) Retinal+ IPTG+ Azide+ Light+

Colony Forming Units

DH5α Controls

IPTG Cytotoxicity proved

Cytotoxic effects of various inputs

Effectors of Growth Rate

The least concentration of Dextrose (0.2g/l) show a negligible growth rate. Thus, it can be used in further experiments as a selection pressure.

0.5 1 1.5 2 2.5 1 2 3 4 5 6 7 8 9 Cell concentration (g/l) Time (Hour) LB 0.2 g/l 0.4 g/l 1 g/l 3 g/l 5 g/l

Growth Profiles based on different Dextrose Concentrations

0.2 0.4 0.6 0.8 1 5 10 15 Cell Conc (g/l) Time (hr)

Growth Profile : LB vs M9 Media (IPTG Induced)

pET41a+PR(IPTG Induced) in Minimal Media pET41a+PR (IPTG Induced) in LB

Experiments

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

• f specific growth rate

IPTG Induced PR+pET41a in LB PR+pET41a Control in LB 0.5 1 1.5 2 2.5 3 2 4 6 8 10 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 0.2 M9 Media control

Project Artemis - Design

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 RFP GPR

Absorption Spectrum Wavelength

0.5 1 1.5 2 2.5 2 4 6 8 OD600 Time (Hours)

Growth Profile

0.02 0.1 0.5 LB LB+0.2 100 200 300 400 500 600 700 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

PcstA promoter characterization

P S X E

BBa_K572200

Proteorhodopsin

BBa_K572005

Backbone of pSB1C3, without Chloramphenicol Resistance Gene

pSB1Pe – Expression Vector

PcstA

Milestones

Carbon stress expression indicator (BBa_K572001) characterized and works as expected Proteorhodopsin coding gene (BBa_K572005) modified to Parts Registry Standard 10, characterized and works as expected. Beta-carotene dioxygenase (BBa_K572009) modified to Parts Registry standard 10 and submitted. Improved and characterized existing part PcstA promoter (BBa_K118011) activity

Milestones

Created software tool for generating Site Directed Mutagenesis primers for silent mutation of Parts Registry Standard restriction enzyme sites from coding sequence Designed a computational model and validated that PR confers metabolic advantage to cells under carbon stress. Designed pSB1Pe & pSB1Pc - plasmids with PR selectivity marker

Way Ahead

• Characterize plasmid pSB1PC and pSB1PE for media with

different carbon substrates.

• Proteorhodopsin in B.subtilis and Agrobacterium sp for
• Hyaluronic acid Production
• Curdlan production
• Anaerobic phased solid digestor for PR expressing bacteria
• Characterize Blue Light Absorbing PR

References

• 1. Walter et al 2006, “Light empowering Escherichia coli with proteorhodpsin”
• 2. Martinez et al 2006, “Proteorhodopsin photosystem gene expression enables

photophosphorylation in a heterologous host”

• 3. Jagannath et al 2009,”Influence of competing metabolic processes on the molecular weight of

hyaluronic acidsynthesized by Streptococcus zooepidemicus”

• 4. Yu et al 2007,”Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid”
• 5. Adam M Feist et. al "A genome-scale metabolic reconstruction for Escherichia coli K-12

MG1655 that accounts for 1260 ORFs and thermodynamic information"

Acknowledgement

• 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 for their financial support

Acknowledgement

Advisors :

• Dr. G K Suraishkumar and DR. Nitish R

Mahapatra of Department of Biotechnology IIT Madras

Thank You!