New Interdisciplinary Approaches to the Engineering of Biology
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Transcript of New Interdisciplinary Approaches to the Engineering of Biology
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
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
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 )
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
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
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
Microsystem-Based Devices for Studying Single Cells
Medium flow
Additions
Microscope Objective
Chemical sensors
To analysischamber
ProteomicsRT-PCR
Fluorescentreporters
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
Measure Gene Expression in Real Time
Promoter fusions with fluorescent proteinsCan measure up to 9 different colors (10 nm apart)
T. Strovas
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
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
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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
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)
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
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Single Cell Division Times
Time, Hr
Single Cell Division Times During MeOH Growth
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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
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