Microfluidics: Physics and Biology

Part III: Microfluidics for Microbes

Jeffrey Moffitt Erel Levine Ronen Kopito

July 13, 2012 Harvard University Center for Systems Biology

Yale University Physics of Living Systems

Topics for Today

Single-cell Questions Technical Limitations

Using more biologically friendly materials

Case Examples

Introduction What scientific questions require the

study of single cells?

What are the limitations of current methods?

How do Cells Make Decisions?

David Goodsell

Environmental Stimuli Antibiotic Stress

Starvation

+

Single-Cell Heterogeneity

Stimulus

Bacterial Culture

Heterogeneous Response

=

Limits of Current Methodology Place on supportive media Observe

A little later…

Unchecked growth overwhelms gel

Limited Observation and Control

Restricted Observation Duration 1. Heterogeneity and rare phenotypes 2.  Inheritance

Restricted Chemical and Spatial Control 3.  Cellular response 4.  Intracellular interaction

Solution: Microfluidics!

Case Example 1: Rare Phenotypes How to study the one in a million

cells that survive antibiotic treatment?

The Persister Cell

Balaban et al., Science 2004 Lewis, Ann. Rev. Microbio., 2010

?

A Microfluidics Solution

Balaban et al., Science 2004

Persisters Precede Antibiotics

Persisters are not growing before ampicillin treatment!

Case Example 2: Inheritance Do bacteria age?

Do Bacteria Age?

Do symmetric bacteria age? Multicellular organisms age

Some unicellular organisms age Ackermann et al., Science 2003

Not Quite Symmetric Division

The Mother Machine

Wang et al., Current Bio. 2010

Aging of E. coli

E. coli age!

Case Example III: Intercellular communication

Can Biologically Friendly Materials Replace PDMS?

Can one create controlled environments to study intercellular

communication?

Moffitt, Lee, and Cluzel, Lab Chip, 2012

Polydimethylsiloxane PDMS

http://drajput.com/research/sylgard184-pdms.php http://www.jove.com/video/319/pdms-device-fabrication-and-surface- modification

Qin & Whitesides, Nature Protocols, 2010

Drawbacks of PDMS

~ 20 microns! PDMS is firm, and bacteria are sensitive to pressure!

PDMS is non permeable to water, restricting nutrient delivery

PDMS can be toxic! Mather et al., PRL 2011 Cho et al., PloS Bio 2007

Mannick et al., PNAS 2010 Regeher et al., Lab Chip 2009

The Best of Both Worlds

Sub-micron tracks to direct cell growth

Large gutters to wash away excess cells

Device 1 How not to build a single-cell chemostat

Creation of Si Negative

10 microns

1.5 micron deep 0.6, 0.7, 0.8 microns wide

50/100 microns

Printing Agarose

Add Molten Gel Cast at 30C

3% w/v Low Melt Agarose Gel SEM of Si Master

4 mm

Introducing Buffer Flow

Growth of Linear Colonies

Not enough flow!

~1 uL/min Δp ~ 2-3 atm

Device Number 2 How to build a single-cell chemostat

Device Number 6 How to build a single-cell chemostat

Increasing Gutter Size

10x10 microns 100x40 microns

First etch tracks

Then etch gutters 40 microns

100 microns

Printing Sub-Micron Features

Silicon Positive

1 cm

PDMS Negative 3-5% Agarose Gel

4 mm 1 cm

Growth on Patterned Agarose

Phase Contrast 1.49NA; 1 min time lapse mg1655; Ampicillin; 5% Low Melt Agarose

Is Growth Perturbed?

Are features E. coli specific?

4% Low Melt Agarose Phase Contrast 1.49NA; 1 min time-lapse

B. subtilis, 3610 in LB E. faecalis, E1sol in BHI

Soft agarose deforms to different cell morphologies!

Cell-Cell Communication

Agarose is porous: diffusive communication is possible

Wintermute & Silver, Genes & Dev. 2010 D’Onfrio et al, Chem. & Bio., 2010

Complementing Auxotrophs

ΔargC

ΔilvE ΔargC

ΔilvE

ΔilvE

4.5% Low Melt Agarose Phase Contrast 1.49NA; 300 nm by 50-µm tracks

Is E. coli Naturally Charitable?

Auxotrophic stress induces the sharing of amino acids

wt wt

ilvE wt

4.5% Low Melt Agarose; Phase Contrast 1.49NA; 5 min time-lapse

Summary and Conclusions

Microfluidic Possibilities Important dynamics happen at the single- cell level Microfluidics allow …

The study of rare phenotypes

Observation of long term inheritance

Spatial and temporal control at the micron scale!

Questions and Demonstration