Microfluidic Single-Cell Analysis for Bioprocess Development

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1 Microfluidic Single-Cell Analysis for Bioprocess Development 03 April 2014 Dietrich Kohlheyer Microscale Bioengineering Group IBG-1: Biotechnology Forschungszentrum Jülich GmbH

2 Population Heterogeneity in Microbial Production Processes Single-cell analysis & synthetic biology What s really going on at single cell-level? cells / L Product [mmol / L] Conventional analytics deliver Averaged information! 2

3 Addressing single cells IBG-1 technology portfolio: Biosensors FACS Time-lapse imaging Image analysis µ-fluidics Versatile sensing applications High-throughput Snapshot High spatiotemporal resolution Cell tracking Lineage analysis Environmental control 3

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5 Single - Use Polydimethylsiloxan (PDMS) Microfluidic Chips for Bacteria Cultivation 5 5

6 Photolithography based HNF Jülich *not drawn to scale Grünberger A, Probst C, Heyer A, Wiechert W, Frunzke J, Kohlheyer D (2013) Journal of visualized experiments: DOI: /

7 Rapid Silicone Molding of Microstructures *not drawn to scale Time to chip < 4 weeks 7

8 Large Scale vs. Micro Scale Bioreactor Microtiter plates Microfluidic chip m³-ml ml - µl nl - fl turbulent / gradients Cells change environment Constant environment Laminar / well predictable 8 8

9 Platform technology for diverse microorganisms Bacteria Archaea Yeast Cells Micro algae Fungi 9

10 Automated Image Analysis Modeling and Simulation Group, Dr. Katharina Nöh 10

11 Microbial Single-Cell Analysis: Fields of application Grünberger A, Wiechert W, Kohlheyer D, Current Opinion in Biotechnology

12 Growth studies of C. Glutamicum at pl scale µ = 0.39h-1 ± 0.03 µ = 0.42h-1 ± 0.01 C. Glutamicum WT, CGXII medium, 30 C µ = 0.44h-1 ± 0.03 µ = 0.63h-1 ± x faster. Grünberger A, van Ooyen J, Paczia N, Rohe P, Schiendzielorz G, Eggeling L, Wiechert W, Kohlheyer D, Noack S (2013) Biotechnology and Bioengineering 110: Unthan S, Grünberger A, van Ooyen J, Gätgens J, Heinrich J, Paczia N, Wiechert W, Kohlheyer D, Noack S (2013). Biotechnology and Bioengineering

13 Growth studies of C. Glutamicum at pl scale BHI Glucose Gluconate Fructose Pyruvate Lactate Acetate Citrate Succinate Malate Without main C-source C. Glutamicum WT, continuous supply of adapted CGXII media, 30 C 13 13

14 Producer Strain characterization in continuous mode C. Glutamicum L-arginine producer Schendzielorz G, Dippong M, Grünberger A, Kohlheyer D, Yoshida A, Binder S, Nishiyama C, Nishiyama M, Bott M, Eggeling L ACS Synthetic Biology (1)

15 Optogenetic Sensors for HT Production Strain Analysis Stephan Binder Georg Schendzielorz IBG-1, Forschungszentrum Jülich GmbH Small-molecule sensors enable high-throughput screening & isolation of microbial producer cells

16 Sensor Strain characterization at defined environments Corynebacterium glutamicum strains carrying psenlys-spc (based on LysG) Arginine reporter fluorescence triggered by di-peptide perfusion Schendzielorz G, Dippong M, Grünberger A, Kohlheyer D, Yoshida A, Binder S, Nishiyama C, Nishiyama M, Bott M, Eggeling L ACS Synthetic Biology (1)

17 Summary and Outlook towards complex m³ bioreactor picoliter m³ emulation pl strain optimization analysis 10 µm 10 µm Innovation & Integration: Temperature fluctuations Gas fluctuations Concentration gradients Complex flow pattern Current state: Versatile platform technology HNF Jülich Short innovation cycles Constant culture environments 17

18 Acknowledgements Alex, Christopher, Christina, Phillip, Agnes, Johanna, Nadja, Stefan, Christian, Julio, Christian, Camille, Sophie Helmholtz Nanoelectric Facility Groups of Julia Frunzke Lothar Eggeling Thomas Drepper Stephan Noack Katharina Nöh Eric von Lieres Martina Pohl Wolfgang Wiechert