1. Real-Time Multivariate Detection from Single Cells Monitoring the Metabolism of Methylobacterium extorquens AM1

2. Overview Microscale Life Science Center Methylobacterium extorquens AM1 Green Fluorescent Protein (GFP) as a transcriptional reporter Detection of respiration rates Multi-variate detection of single cells

3. MLSC Funded by NIH CEGS To develop technologies for single cell research Lab-on-a-chip modality

4. Why Single Cells? Variable of interest Bulk data represents averages Averages may not represent behavior of subpopulations

5. Methylobacterium extorquens AM1 Gram - bacterium (like E. coli) Capable of growing on methanol and multicarbon substrates (succinate) Industrial interest for production of value added products

6. periplasm cytoplasm MeOH Methylotrophic Metabolism Formaldehyde Central Metabolism (Methanol Dehydrogenase) (Formaldehyde Activating Enzyme) (Carbon Assimilation)

7. Goals Hypothesis: Behavior of single cells differ from that of averaged populations Approach: Develop and utilize technology to study single cells Characterize single cells in contrast to populations

8. Populations to Single Cells Use GFP as a reporter of transcriptional activity Will reflect promoter activity Observed GFP fluorescence during growth on methanol and succinate Observe in bulk and at the single cell level

9. Green Fluorescent Protein First isolated from Aequorea victoria Emits fluorescence at 509nm Coral is another source for many color variants

10. Genetic Manipulation Suicide Vector Chromosome KanRGFPuv Double Crossover Event Red regions = homologous sequence

11. Genetic Fusions P MDH GFPuv Transcriptional Fusion Methanol Growth Higher GFP expression Succinate Growth Lower GFP expression

12. Fluorimetry Strovas et al. In preparation.

13. Data can be used for the calculation of promoter activities Is a gauge of gene transcription in bulk culture Promoter activity dictated by multiple variables Calculating Promoter Activities

14. Equations for Modeling Promoter Activity Leveau and Lindow, 2001 Non-fluorescent FP (n) Fluorescent FP (f) Dilution from Cell Division Degradation Maturation Synthesis P m n  n  f V max n n + f + K M V max f n + f + K M

15. Establish  RFU/  O.D. 600nm plot P = f ss *  (1 +  /m) f ss =  RFU/  OD 600nm  = generation time m = maturation rate of GFP Units are RLU/OD 600nm *hr Equations for Modeling Promoter Activity

16. Fluorimetry Strovas et al. In preparation. 349.1 +/- 82.59 264.3 +/- 10.27

17. Single Cell Growth Assays Observed growth of single cells Determined divisions rates Measured fluorescence content

18. Single Cell Growth Assays Video using LSM software

19. LSM Experiments Strovas et al. In preparation.

20. LSM Experiments Strovas et al. In preparation. 0.55  m/hr 0.73  m/hr

21. LSM Experiments Strovas et al. In preparation. 3.12 +/- 0.55 hrs (N = 115)

22. LSM Experiments Strovas et al. In preparation. 3.73 +/- 0.63 hrs (N = 195)

23. LSM Experiments Single Cell Growth on Succinate Strovas et al. In preparation.

24. LSM Experiments Single Cell Growth on Methanol Strovas et al. In preparation.

25. LSM Experiments Single Cell Carbon Shifts Succinate: 1993.15 +/- 468.14 RFU/  m^2 (N = ~1000) Methanol: 3075.30 +/- 243.35 RFU/  m^2 (N = ~1000) Strovas et al. In preparation.

26. GFPuv is a viable reporter in M. extorquens AM1 Data averages obscure subpopulation dynamics Populations to Single Cells

27. Measuring Respiration Rates Measured respiration rates from bulk cultures of M. extorquens AM1 Utilized Pt-porphyrin doped beads that are an inverse sensor of [O 2 ] Signals acquired are phosphorescent lifetimes Samples and beads were sealed in 4ml cuvette and monitored over time

28. Fluorescence Phosphorescence Intersystem crossing Absorption Quenching O2O2 Energy Singlet Excited State Triplet Excited State Bulk Respiration rates

29. LightDark I o (1 – e -Kt )I o e -Kt a b Log(b/a) = Lifetime of decay Bulk Respiration rates

30. Strovas and Dragavon et al. J. Environ Microbiol. (accepted)

31. Bulk Respiration rates Strovas and Dragavon et al. J. Environ Microbiol. (accepted) Respiration rate (Mol O/min*cell e-17) Methanol = 5.4 +/- 0.74 Succinate = 3.8 +/- 0.89

32. Multi-variate detection from single cells Utilize multiple fluorescent proteins as transcriptional probes Measure respiration rates as a gauge of metabolic activity and cell health

33. Methylotrophic Metabolism GFP YFP RFP Methanol Oxidation Formaldehyde Oxidation Carbon Assimilation Central Metabolism

34. Current Approach Aqueous phase Hydrophobic Phase Oil water separation for spatial isolation Utilize 50-100  m square capillaries Use free floating porphyrin beads

35. Oil and Water 250  m capillary 4nL aqueous volumes

36. End Goals Achieve single respiration rate detection Measure gene expression in single cells with three fluorescent proteins Use all four measurements as a comprehensive analysis of M. extorquens AM1 response to growth on methanol and succinate

37. Acknowledgements Dr. Mary Lidstrom MLSC The Lidstrom Lab Dr. Joseph Chao Dr. Mark Holl Joe Dragavon Tim Molter Cody Young Linda Sauter Tylor Hankins Angela Burnside