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.