Real-Time Multivariate Detection from Single Cells

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

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 Aequoreavictoria • Emits fluorescence at 509nm • Coral is another source for many color variants

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

11. PMDH GFPuv Genetic Fusions Transcriptional Fusion • Methanol Growth Higher GFP expression • Succinate Growth Lower GFP expression

12. Fluorimetry Strovas et al. In preparation.

13. Calculating Promoter Activities • 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

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

15. Equations for Modeling Promoter Activity • Establish dRFU/dO.D. 600nm plot • P = fss*m(1 + m/m) • fss = dRFU/dOD600nm • m = generation time • m = maturation rate of GFP • Units are RLU/OD600nm*hr

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

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 0.55mm/hr 0.73 mm/hr Strovas et al. In preparation.

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

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

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/mm^2 (N = ~1000) Methanol: 3075.30 +/- 243.35 RFU/mm^2 (N = ~1000) Strovas et al. In preparation.

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

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 [O2] • Signals acquired are phosphorescent lifetimes • Samples and beads were sealed in 4ml cuvette and monitored over time

28. Singlet Excited State Triplet Excited State O2 Intersystem crossing Quenching Energy Absorption Fluorescence Phosphorescence Bulk Respiration rates

29. Light Dark Io(1 – e-Kt) Ioe-Kt a b Log(b/a) = Lifetime of decay Bulk Respiration rates

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

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

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 Methanol Oxidation YFP Formaldehyde Oxidation Carbon Assimilation RFP Central Metabolism

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

35. Oil and Water 250mm 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. Dr. Mary Lidstrom MLSC The Lidstrom Lab Acknowledgements • Dr. Joseph Chao • Dr. Mark Holl • Joe Dragavon • Tim Molter • Cody Young • Linda Sauter • Tylor Hankins • Angela Burnside