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New Interdisciplinary Approaches New Interdisciplinary Approaches to the Engineering of Biologyto the Engineering of Biology
Combine Combine •GenomicsGenomics•Computational biologyComputational biology•MEMS (microelectromechanical systems)MEMS (microelectromechanical systems)•Systems integrationSystems integration•NanotechnologyNanotechnology
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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
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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 )
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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
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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
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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
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Microsystem-Based Devices for Studying Single Cells
Medium flow
Additions
Microscope Objective
Chemical sensors
To analysischamber
ProteomicsRT-PCR
Fluorescentreporters
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System Setup with Laser Scanning Confocal Microscope in the MLSC
Overview of Setup
Andor CCD Camera
Laser Scanning Microscope
Mini-environmental Chamber
EnvironmentControl Devices
Multiwavelength fluorescenceTemperature controlMedium flow-through
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Measure Gene Expression in Real Time
Promoter fusions with fluorescent proteinsCan measure up to 9 different colors (10 nm apart)
T. Strovas
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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
Applied as a Paint
Applied Photolitho-graphically
Incorporated into a
Polystyrene Matrix
Dendrimer Solution
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O2 Consumption Sensor for Single Cells
platinum-porphryin compound imbedded in beads (1m)
Calibration of Sensor Response to Dissolved Oxygen
Concentrations
y = 5.8099x
R
2
= 0.9905
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0% 5% 10% 15% 20%
Percent Oxygen
((I
o
/I)-1
)
Bacterial Oxygen Consumption in a Closed System
0.00%
5.00%
10.00%
15.00%
20.00%
0 10 20 30 40 50
Time (min)
% Dissolved Oxygen
A B 10 cells/nl
T. Strovas, T. Hankins, J. Callis, M. Holl, D. Meldrum
A B C
21%O2 5% O2
beads
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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)
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Evidence for Heterogeneity• Single-cell cell cycle analysis: growth
Tim Strovas,
Linda Sauter 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3
# cells
0
2
4
6
8
10
12
Single Cell Division Times
Time, Hr
Single Cell Division Times During MeOH Growth
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63
Time (hrs)
Range:2.5-4.3 hr
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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