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 Applications BioEnergy

E. coli that resists mutations (Alex)Cellular Computation (George)Microfluidics (George)DNA transfer using virus (Shaunak)Combinatorial Constructions and SelectionsBioBricks (Nick)

Producing a Renewable Energy SourceCellulose 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 SourceLight 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 RBS RBS

Pathway / Metabolic EngineeringMicrobial 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 EngineeringMicrobial Therapy, BioEnergy

Optimization

conversion of six molecules of xylose into 10 molecules each of ethanol and CO2 J Bacteriol. 2001 May; 183(10): 2979–2988

glycolysis

Clone or Make SyntheticallyRBS 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

RBS

SensorsBiosensor, Quorum Sensing, BioEnergy

RBS Sensor Response 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 measureSense Deficiency -> Trigger Synthesis of vitaminSense Arsenic -> Detectable output or

Trigger SequestrationTriggered “Kill Switch” for safety

Optimization

Cellular TargetingBacterial Surface Display and Aptamers

(Potential Applications)

Bind Proteins

Bind Other Cells

Bind Tissue Types

Bind Surface

Bind DNA

Bind Viruses

Cell-cell InteractionsCell-cell targeting

Tissue Targeting

Streptavidin

Microscale patterning

Combinatorial Constructions and Selections – Many New BioBricks

Bind Toxins

Fusion Protein

Want to Coat Bacteria withProteins of Interest

Surface Display: Fusions to Membrane Proteins

StreptavidinStrep Binding PeptidesHistidine TagRandom 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. 2000 Apr 28;298(2):273-82.

OmpA Outer Membrane Protein

LlpLeader OmpA

BioBrick Insertion SiteLlp

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.

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. 2004 Mar 24;23(6):1257-66. Epub 2004 Mar 11.

NalP Autotransporter

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.

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. 2006 Jul;188(13):4841-50.