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Microbial Therapy (Steph, Alex, Sammy) Pathway Engineering

Brainstorming Session Overview : Useful Applications of Synthetic Biology. Medical / Human Applications. BioEnergy. Microbial Therapy (Steph, Alex, Sammy) Pathway Engineering - make product body needs (possibly sense deficiency) - Synthetic Symbiosis

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Microbial Therapy (Steph, Alex, Sammy) Pathway Engineering

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  1. Brainstorming Session Overview: Useful Applications of Synthetic Biology Medical / Human Applications BioEnergy Microbial Therapy (Steph, Alex, Sammy) Pathway Engineering - make product body needs (possibly sense deficiency) - Synthetic Symbiosis (E. coli natural gut environment) - Vitamin B Production (Steph and Alex) - Fibrin (clotting) (Sammy) - Insulin (Harris) - Degrade Plaque - Implantable Devices that release or synthesize drugs - Off-switch for safety, competitive concerns (Alex) Cellular Targeting - Aptamers (Steph) - Bacterial Surface Display / Fusion Prot. (Perry) - Target Bact to certain tissues (actuator or product) - Targeting Microbial Factories - Sequestration of Toxic Compounds (Sammy) Bacterial Biosensors - Sense and Sequester Toxic Compounds (arsenic) (Sammy) - Quorum Sensing - Radon Sensor Biofilms and Vascularization (Harris and Sammy) Recombination (Harris) Producing a Renewable Energy Source Cellulose to EtOH (Shaunak, Steph, Alex) Fatty Acid production and degradation for energy Synthesis, Tolerance, and Export of EtOH or alkanes Alternative organisms for Energy Prod. Yeast (Shaunak) Bacteria as Energy Source Light Powering E. coli w/ Photorhodopsin (Alex) Powering Implantable Medical Devices Light sensitive Proton Pump (Pseud. Putida) Biological Based Fuel Cells (Sammy) Bridging Applications E. coli that resists mutations (Alex) Cellular Computation (George) Microfluidics (George) DNA transfer using virus (Shaunak) Combinatorial Constructions and Selections BioBricks (Nick)

  2. Pathway / Metabolic Engineering Microbial Therapy, BioEnergy Input (Import) Set of genes (reasonable #) Output / Product (Export) Assay Source/ Availability Optimization Clone or Make Synthetically RBS RBS RBS Promoter Cellulose to EtOH -> Sugar (Xylose) to EtOH (pdc and adhB genes, L. Ingram) pyruvate decarboxylase, alcohol dehydrogenase II Vitamin Production (operon structure) Natural Product Production (Fibrin, Insulin) Plaque Degradation Synthesis of an antibiotic Fatty Acid Production or alkanes Could couple to a sensor (make when needed) Use BioBrick Strategy Artemisinin example, Malaria Drug, Keasling Lab

  3. Pathway / Metabolic Engineering Microbial Therapy, BioEnergy Input (Import) Set of genes (reasonable #) Output / Product (Export) Assay Source/ Availability Optimization Clone or Make Synthetically RBS RBS RBS Promoter conversion of six molecules of xylose into 10 molecules each of ethanol and CO2 J Bacteriol. 2001 May; 183(10): 2979–2988 Cellulose to EtOH -> Sugar (Xylose) to EtOH (pdc and adhB genes, L. Ingram) pyruvate decarboxylase, alcohol dehydrogenase II Vitamin Production (operon structure) Natural Product Production (Fibrin, Insulin) Plaque Degradation Synthesis of an antibiotic Fatty Acid Production or alkanes Could couple to a sensor (make when needed) Use BioBrick Strategy glycolysis

  4. Sensors Biosensor, Quorum Sensing, BioEnergy Sensor Response Element RBS RBS Assay Optimization Sensors -respond to external commands -Can be used to turn genes on and off Sense EtOH buildup -> Trigger protective measure Sense Deficiency -> Trigger Synthesis of vitamin Sense Arsenic -> Detectable output or Trigger Sequestration Triggered “Kill Switch” for safety 1. Cytoplasmic Regulatory Proteins 2. Two-Component Systems 3. Environment Responsive Promoter 4. Regulatory RNAs

  5. Cellular Targeting Bacterial Surface Display and Aptamers (Potential Applications) Bind Viruses Bind Toxins Bind Surface Bind Proteins Microscale patterning Bind Other Cells Bind DNA Bind Tissue Types Streptavidin Cell-cell Interactions Cell-cell targeting Tissue Targeting Combinatorial Constructions and Selections – Many New BioBricks

  6. Cellular Targeting Surface Display: Fusions to Membrane Proteins Fusion Protein OmpA Want to Coat Bacteria with Proteins of Interest Autotransporters Streptavidin Strep Binding Peptides Histidine Tag Random Library (Peptides)

  7. Surface Display: OmpA – Outer Membrane Protein A Llp Leader OmpA BioBrick Insertion Site Llp Leader 2 OmpA BioBrick Insertion Site Georgiou G, Stephens DL, Stathopoulos C, Poetschke HL, Mendenhall J, Earhart CF. Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions. Protein Eng. 1996 Feb;9(2):239-47. Pautsch A, Schulz GE. High-resolution structure of the OmpA membrane domain. J Mol Biol. 2000 Apr 28;298(2):273-82. OmpA Outer Membrane Protein

  8. Surface Display: Autotransporter System: AIDA-1 Leader Sequence Linker Autotransporter BioBrick Insertion Site Jose J.Autodisplay: efficient bacterial surface display of recombinant proteins. Appl Microbiol Biotechnol. 2006 Feb;69(6):607-14. Epub 2005 Dec 20. Review. Maurer J, Jose J, Meyer TF. J Bacteriol. 1997 Feb;179(3):794-804. Oomen CJ, van Ulsen P, van Gelder P, Feijen M, Tommassen J, Gros P. Structure of the translocator domain of a bacterial autotransporter.EMBO J. 2004 Mar 24;23(6):1257-66. Epub 2004 Mar 11. NalP Autotransporter Jain S, van Ulsen P, Benz I, Schmidt MA, Fernandez R, Tommassen J, Goldberg MB. Polar localization of the autotransporter family of large bacterial virulence proteins. J Bacteriol. 2006 Jul;188(13):4841-50.

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