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

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

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

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

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