1. New Interdisciplinary Approaches to the Engineering of Biology Combine GenomicsGenomics Computational biologyComputational biology MEMS (microelectromechanical systems)MEMS (microelectromechanical systems) Systems integrationSystems integration NanotechnologyNanotechnology

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

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

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

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. Measure O 2 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 Applied as a Paint Applied Photolitho- graphically Incorporated into a Polystyrene Matrix Dendrimer Solution

11. O 2 Consumption Sensor for Single Cells platinum-porphryin compound imbedded in beads (1  m) A B 10 cells/nl T. Strovas, T. Hankins, J. Callis, M. Holl, D. Meldrum A B C 21%O 2 5% O 2 beads

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

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

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