1. 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)

2. 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)

3. EECE 491c Balagaddé et al. (2005) Science 309:137-140 3 Scaling Bioreactors Down to Nanoliters… 1 1x10 -3 1x10 -6 1x10 -9 1x10 3 1x10 6 1x10 9 Liters Advantages: Reduced reagent costs High-throughput experiments Cell tracking Reduced waste Increased speed of experiments

4. EECE 491c Balagaddé et al. (2005) Science 309:137-140 4 How do we culture cells? Batch Fed-batch (semi continuous) Chemostat (continuous) Time Number of bacteria

5. EECE 491c Balagaddé et al. (2005) Science 309:137-140 5 Bacteria and biofilms (www.medicdirect.co.uk)

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

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

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

9. EECE 491c Balagaddé et al. (2005) Science 309:137-140 9 The Microchemostat ExperimentMedium [ glucose] (M) [bacto- tryptone] (g/L) Temperature ( o C) Dilutio n rate (h -1 ) 1MOPS EZ1.1--320.34 2MOPS EZ0.11--320.30 3LB--0.5210.24 4LB--0.5210.30 5LB--3.0210.37 6LB--0.5210.37 7LB--0.1210.37

10. EECE 491c Balagaddé et al. (2005) Science 309:137-140 10 The Microchemostat 3 g/L 0.5 g/L 0.1 g/L

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

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

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

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

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

16. EECE 491c Balagaddé et al. (2005) Science 309:137-140 16 MC4100Z1 Cells

17. EECE 491c Balagaddé et al. (2005) Science 309:137-140 17 Top10F’ Cells

18. EECE 491c Balagaddé et al. (2005) Science 309:137-140 18 Loss of Regulation MC4100Z1 Top10F’

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

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

21. 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 10 5 and prolong monitoring of homogeneous populations. Monitoring the programmed behavior of bacterial population for hundred of hours despite strong selection pressure.

22. EECE 491c Balagaddé et al. (2005) Science 309:137-140 22 Critique Summary To be improvedGood Major points Minor points 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