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

P hotonivorous Bacteria for R esolution and E nhanced e X pression Team: IIT Madras Indian Institute of Technology Madras

Selection Markers for Screening Antibiotic Metabolic Resistance Markers Negative selection Positive selection markers markers More risky horizontal Limited strains of gene transfer cells that can be used

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

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

H+ ATP Synthase Electron Transport Chain Glycolysis Carbon Source

Photophosphorylation to the rescue H+ H+ H+ H+ H+ H+ P P P R R R ATP Synthase Electron Transport Chain + Retinal Glycolysis ATP Carbon Source

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

Minimal Media with PR H+ H+ H+ H+ H+ H+ P P P R R R ATP Synthase Electron Transport Chain + Retinal Glycolysis ATP Carbon Source

Recombinant protein production in Minimal Media H+ H+ H+ H+ Recombinant Protein ATP Synthase Electron Transport Chain Glycolysis ATP Carbon Source

Recombinant protein production in Minimal Media H+ H+ H+ H+ H+ H+ Recombinant Protein P P P R R R ATP Synthase Electron Transport Chain + Retinal Glycolysis ATP Carbon Source

SunScreen – Light based Screening of Clones Minimal Media (containing Retinal)

Project Sunscreen - Design Proteorhodopsin Coding Gene BBa_K572005 BBa_K572006 Proteorhodopsin Generator BBa_B0034 BBa_K572005 BBa_B0015 BBa_K572007 IPTG induced Proteorhodopsin Generator T7 Promoter BBa_B0034 BBa_K572005 BBa_B0015 BBa_K572008 Constitutive Proteorhodopsin Generator BBa_J23119 BBa_B0034 BBa_K572005 BBa_B0015

PRIMER INPUTS OUTPUTS • Protein sequence • Primer Sequence • Number and positions • Tm, %GC and of Mutations Length • Range of Tm, %GC and Length of primers

Characterization of Proteorhodopsin K572005 Over Expression of GPR in BL21DE3 Azide Test on BL21 with PR 1 . Relative number of 0.8 Azide Negative CFUs 0.6 Samples 0.4 Azide Treated 0.2 Samples 0 28 kDA IPTG+ Light- IPTG+ Light+ Time after induction 15% SDS PAGE gel showing the IPTG induction of PR expression

Project Sunscreen - Design BBa_K572009 BBa_B0034 BBa_K572009 BBa_B0015 Mouse Beta-Carotene Dioxygenase Generator Dioxygenase Coding Gene Constitutive Dioxygenase generator BBa_J23119 BBa_B0034 BBa_K572009 BBa_B0015 BBa_K274210: Endogenous CrtEBIY synthesis of beta-carotene BBa_K572009 : Retinal Mouse Beta Carotene Biosynthesis Dioxygenase converts beta carotene to Trans-Retinal Retinal with PR in presence of light helps in development of proton gradient β -Diox

pSB1Pc – Cloning Vector Backbone of pSB1C3, without Chloramphenicol Resistance Gene E X S P BBa_K572100 BBa_K572005 Proteorhodopsin

Model - Design Hypothesis: Increase in growth rate due to Proteorhodopsin proton efflux in minimal carbon media H + H + ATP Synthase Proteorhodopsin H + H + Genomic Scale Metabolic Pathway *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 ”

Metabolic Modeling Hypothesis: Increase in growth rate due to Proteorhodopsin proton efflux in minimal carbon media • Network Composition (*, **) • Reaction and Specie parameters SBML • SBML toolbox, MATLAB • Setting up model in COBRA (readCbModel) COBRA Model • Model validated using literature data Analysis • Growth rate variation from WT to PR *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 ”

Model Simulations for Validations Glucose Growth Rate Growth Rate Uptake flux (hr-1) (hr-1) ( mmol/g-dw hr) Model_WT Model_PR Complete Inhibition (Azide) 2 0 0 3 0 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 to find minimal media Glucose uptake Rate (mmol / g-dw hr) model_WT model_PR 0.35 0 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 Cytotoxic effects of various inputs DH5 α Controls Colony Forming Units 50 40 30 20 10 0 Global Retinal+ IPTG+ Azide+ Light+ Negative (R-I-A-L-)  IPTG Cytotoxicity proved

Effectors of Growth Rate Growth Profiles based on different Dextrose Concentrations 2.5 The least concentration of Dextrose (0.2g/l) show a negligible growth rate. Thus, 2 it can be used in further Cell concentration (g/l) experiments as a selection LB 1.5 pressure. 0.2 g/l 0.4 g/l 1 1 g/l 3 g/l 5 g/l 0.5 0 0 1 2 3 4 5 6 7 8 9 Time (Hour)

Experiments Comparison of rate of change Growth Profile : LB vs M9 Media of specific growth rate (IPTG Induced) 3 Specific growth rate (Hour -1) 1 2.5 0.8 Cell Conc (g/l) 2 pET41a+PR(IPTG 0.6 1.5 Induced) in Minimal 1 Media 0.4 pET41a+PR (IPTG 0.5 0.2 Induced) in LB 0 0 0 2 4 6 8 0 5 10 15 time(hour) Time (hr) IPTG Induced PR+pET41a in LB PR+pET41a Control in LB Comparison of specific growth rate in M9 media 3 Specific Growth rate (Hour-1) 2.5 2 IPTG Induced5g/l M9 media 1.5 5g/l M9 media Control 1 0.2 M9 Media control 0.5 0 0 2 4 6 8 10 Time (Hour)

Project Artemis - Design BBa_K572001 : Carbon Stress Response Indicator BBa_K118011 BBa_K081014/mRFP1 gen (PcstA) Carbon Stress Induced Promoter Absorption Spectrum Substrate Availability GPR RFP Bba_K572001 Time 525 nm 584 nm Wavelength

PcstA promoter characterization Growth Profile RFP Expression 2.5 700 600 2 0 0 500 RLU/OD600 OD600 1.5 0.02 0.02 400 300 0.1 0.1 1 200 0.5 0.5 0.5 100 LB LB 0 0 LB+0.2 LB+0.2 0 2 4 6 8 0 2 4 6 8 Time (Hours) Time (Hours) As viewed under a fluorescence microscope, RFP being expressed in DH5α with PcstA promoter

pSB1Pe – Expression Vector Backbone of pSB1C3, without E Chloramphenicol Resistance Gene X S PcstA P BBa_K572200 BBa_K572005 Proteorhodopsin

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!