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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?
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?
Do Bacteria Age?
Do symmetric bacteria age? Multicellular organisms age
Some unicellular organisms age Ackermann et al., Science 2003
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
Increasing Gutter Size
10x10 microns 100x40 microns
First etch tracks
Then etch gutters 40 microns
100 microns
Growth on Patterned Agarose
Phase Contrast 1.49NA; 1 min time lapse mg1655; Ampicillin; 5% Low Melt Agarose
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
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!