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) Medical / Human ApplicationsBioEnergy E. coli that resists mutations (Alex) Cellular Computation (George) Microfluidics (George) DNA transfer using virus (Shaunak) Combinatorial Constructions and Selections BioBricks (Nick) 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 Brainstorming Session Overview: Useful Applications of Synthetic Biology

Input (Import) Output / Product (Export) AssaySet of genes (reasonable #) Clone or Make Synthetically Source/ Availability RBS Pathway / Metabolic Engineering Microbial Therapy, BioEnergy Promoter Artemisinin example, Malaria Drug, Keasling Lab Optimization 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

Input (Import) Output / Product (Export) AssaySet of genes (reasonable #) Source/ Availability Pathway / Metabolic Engineering Microbial Therapy, BioEnergy Optimization conversion of six molecules of xylose into 10 molecules each of ethanol and CO2 J Bacteriol May; 183(10): 2979–2988 glycolysis Clone or Make Synthetically 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

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

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

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

Surface Display: OmpA – Outer Membrane Protein A Pautsch A, Schulz GE. High-resolution structure of the OmpA membrane domain. J Mol Biol Apr 28;298(2): OmpA Outer Membrane Protein 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 Feb;9(2):

Surface Display: Autotransporter System: AIDA-1 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 Mar 24;23(6): Epub 2004 Mar 11. NalP Autotransporter Jose J. Jose J. Autodisplay: efficient bacterial surface display of recombinant proteins. Appl Microbiol Biotechnol Feb;69(6): Epub 2005 Dec 20. Review. Maurer J, Jose J, Meyer TF. J Bacteriol Feb;179(3): Leader Sequence Linker BioBrick Insertion Site 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 Jul;188(13):