New Interdisciplinary Approaches to the Engineering of Biology

1. New Interdisciplinary Approaches to the Engineering of Biology • Combine • Genomics • Computational biology • MEMS (microelectromechanical systems) • Systems integration • Nanotechnology

2. Study Metabolism in Single Cells • Metabolic studies in averaged populations do not capture the range of metabolic events or heterogeneity in subpopulations • Difficult to study activities of rare cells in mixed populations • Difficult to study multiple metabolic parameters in single cells Need: new technologies to study living individual cells in real time

3. Single Cell Challenges • Volume of a bacterial cell ~ fl (10-15) • Number of DNA molecules ~2-3 • Number of mRNA molecules for a specific gene ~10-10,000 • Total protein amount ~amoles (10-18) • Total moles of specific metabolites ~ amoles (10-18) • Respiration rates ~fmol/min/cell (10-15 )

4. Microscale Life Sciences CenterUniversity of Washington • Center of Excellence of Genomic Sciences funded by NIH NHGRI • Co-directed by Mary Lidstrom and Deirdre Meldrum (EE) • Started August 2001 • Goal: • Study complex processes in individual living cells • Chemists, biologists, engineers working together

5. nl chamber • Microelectromechanical systems (MEMS) • Devices, pumps, syringes, valves, sensors, etc. at the mm scale • Nanoelectromechanical systems (NEMS) How to Analyze Single Cells? • Small volumes • fmol per nanoliter = mM! • Need to work with cells in nl volumes

6. What to Measure? TARGETS • Cell processes • Metabolism • Cell cycle • Protein expression • Gene expression MEASUREMENTS • Cell processes • Respiration • Products/substrates • DNA content • Proteomics • Reporters, RT-PCR Fluorescence

7. Chemical sensors Additions To analysis chamber Fluorescent reporters Proteomics RT-PCR Microscope Objective Microsystem-Based Devices for Studying Single Cells Medium flow

8. System Setup with Laser Scanning Confocal Microscope in the MLSC Multiwavelength fluorescence Temperature control Medium flow-through Overview of Setup Environment Control Devices Laser Scanning Microscope Mini-environmental Chamber Andor CCD Camera

9. Measure Gene Expression in Real Time Promoter fusions with fluorescent proteins Can measure up to 9 different colors (10 nm apart) T. Strovas

10. Incorporated into a Polystyrene Matrix Dendrimer Solution Applied as a Paint Applied Photolitho-graphically Measure O2 Consumption in Single Cells • Approach: Use a platinum porphyrin phosphor embedded in a polymer matrix, the molecule’s phosphorescence is quenched by molecular oxygen • Porphyrin can be used in different forms Phosphorescence Intensity Ratio as a Function of Percent Oxygen

11. B A beads A 21%O2 5% O2 B C O2 Consumption Sensor for Single Cells platinum-porphryin compound imbedded in beads (1 mm) 10 cells/nl T. Strovas, T. Hankins, J. Callis, M. Holl, D. Meldrum

12. Single-cell proteomics (Norm Dovichi, Chemistry) Protein fingerprints by 2D capillary electrophoresis • Single-cell RT-PCR (Kelly FitzGerald, ChemE) mRNA for up to 9 genes Post Real-time Analysis (kill cells)

13. Range: 2.5-4.3 hr Evidence for Heterogeneity • Single-cell cell cycle analysis: growth Tim Strovas, Linda Sauter

14. Future Work • Single-cell proteomics • Single-cell RT-PCR • Integrated system to measure (in real-time) • Expression from 4 genes • Respiration rates • Methanol uptake rates Outcomes Cellular-based, mechanistic understanding of methylotrophy as an interconnected dynamic system Global cellular response, at the individual cell level