Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat

Frederick K. Balagaddé, Lingchong You, Carl L. Hansen, Frances H. Arnold, Stephen R. Quake

Article critique presented by Véronique Lecault

January 10, 2006 EECE 491c

Science, 309:137-140 (2005)

EECE 491c Balagaddé et al. (2005) Science 309:137-140 2

Microfluidics: A Small World with Large Applications…

Protein crystallization

DNA amplification

Electrophoresis

Immunoassays

DNA microarrays

Cell sorting and analysis

(Thorsen et al., 2002)

EECE 491c Balagaddé et al. (2005) Science 309:137-140 3

Scaling Bioreactors Down to Nanoliters…

11x10-31x10-61x10-9 1x103 1x106 1x109 Liters

Advantages:

• Reduced reagent costs

• High-throughput experiments

• Cell tracking

• Reduced waste

• Increased speed of experiments

EECE 491c Balagaddé et al. (2005) Science 309:137-140 4

How do we culture cells?• Batch

• Fed-batch (semi continuous)

• Chemostat (continuous)

Time

Num

ber

of b

acte

ria

EECE 491c Balagaddé et al. (2005) Science 309:137-140 5

Bacteria and biofilms

(www.medicdirect.co.uk)

EECE 491c Balagaddé et al. (2005) Science 309:137-140 6

Bacteria and biofilms

EECE 491c Balagaddé et al. (2005) Science 309:137-140 7

The Microchemostat

EECE 491c Balagaddé et al. (2005) Science 309:137-140 8

The Microchemostat

EECE 491c Balagaddé et al. (2005) Science 309:137-140 9

The MicrochemostatExperiment Medium [ glucose]

(M)[bacto-

tryptone] (g/L)

Temperature (oC)

Dilution rate (h-1)

1 MOPS EZ 1.1 -- 32 0.34

2 MOPS EZ 0.11 -- 32 0.30

3 LB -- 0.5 21 0.24

4 LB -- 0.5 21 0.30

5 LB -- 3.0 21 0.37

6 LB -- 0.5 21 0.37

7 LB -- 0.1 21 0.37

EECE 491c Balagaddé et al. (2005) Science 309:137-140 10

The Microchemostat

3 g/L

0.5 g/L

0.1 g/L

EECE 491c Balagaddé et al. (2005) Science 309:137-140 11

Quorum Sensing

EECE 491c Balagaddé et al. (2005) Science 309:137-140 12

Quorum Sensing

EECE 491c Balagaddé et al. (2005) Science 309:137-140 13

Quorum Sensing

EECE 491c Balagaddé et al. (2005) Science 309:137-140 14

Quorum Sensing

IPTG -

EECE 491c Balagaddé et al. (2005) Science 309:137-140 15

Quorum Sensing

IPTG +

EECE 491c Balagaddé et al. (2005) Science 309:137-140 16

MC4100Z1 Cells

EECE 491c Balagaddé et al. (2005) Science 309:137-140 17

Top10F’ Cells

EECE 491c Balagaddé et al. (2005) Science 309:137-140 18

Loss of Regulation

MC4100Z1

Top10F’

EECE 491c Balagaddé et al. (2005) Science 309:137-140 19

Population dynamics

EECE 491c Balagaddé et al. (2005) Science 309:137-140 20

Population dynamics

EECE 491c Balagaddé et al. (2005) Science 309:137-140 21

Conclusions

• Operation of a miniaturized bioreactor 300 times smaller than the previous microfermentor.

• Automatic culturing and monitoring of populations of 100 to 10000 bacteria with instantaneous cell resolution.

• In theory, the mutation rate should be reduced by a factor of 105 and

prolong monitoring of homogeneous populations.

• Monitoring the programmed behavior of bacterial population for hundred of hours despite strong selection pressure.

EECE 491c Balagaddé et al. (2005) Science 309:137-140 22

Critique Summary

To be improved Good

Ma

jor

po

ints

Min

or

po

ints

•Experiments missing to explain what causes the loss of regulation

•Could fit the model to data in order to estimate the true constants

•Could put more emphasis on the absence of biofilm formation and on the limitations of the chemostat

•Should compare mutation rates to a macroscale chemostat instead of a microscale batch

•Very novel technology

•Attempt to show a possible application

•Honesty of the authors

•Clear representation of the microchemostat technology

•Some figures are not very intuitive to read

•Explanation missing for the morphological behavior

•Incomplete Material and methods section leading to hardly reproducible experiments