Thermo Workshop Mannheim 2017-11-09/10 dr. mark doerr university - PowerPoint PPT Presentation

On the way to Synthetic Biology : Enzyme Cascade Optimisation at the University of Greifswald Robotic Platform LARA including applications of the Cytomat 2 ToS Thermo Workshop Mannheim 2017-11-09/10 dr. mark doerr university greifswald

our robotic platform Laboratory Automation Robotic Assistant

colony picking robot( MD QPix420 ) 4 2017-11-09 dr. mark doerr university greifswald

robotic liquid dispenser ( Thermo multidrop ) f a a a s t ! 5 2017-11-09 dr. mark doerr university greifswald

4 robotic incubators ( cytomats 2 ) 6 2017-11-09 dr. mark doerr university greifswald

robotic centrifuge ( Hettich Rotanta 460 R ) 7 2017-11-09 dr. mark doerr university greifswald

liquid handling robot( Agilent Bravo ) 8 2017-11-09 dr. mark doerr university greifswald

UV/fmuorescence reader units for robots Thermo Varioskan barcode reader BMG Omega Fluorostar 9 2017-11-09 dr. mark doerr university greifswald

central robotic arm ( Thermo/Fanuc F5 ) 10 2017-11-09 dr. mark doerr university greifswald

making robot experiments

Our robotic process n 2 OD n t growth (MTP) induction centrifugation/ harvesting 4 difgerent enzyme classes n AU 10 3 with difgerent R production organisms t lysis assay data analysis/ statistics 12 2017-11-09 dr. mark doerr university greifswald

what do we screen ? Current projects cover: ● New and altered substrate specifjcity of amino transferases ● altered enantio-selectivity ● improvement of enzyme stability ( BMVO s) ● improvement of enzyme solubility ( pig liver esterase ) ● de-novo enzyme studies ( AlleyCat ) ● in-vitro-expression of enzymes ( PURE system ) 13 2017-11-09 dr. mark doerr university greifswald

Sandy Schmidt (TU Graz) synthetic biology applications: enzyme cascades 14 2017-11-09 dr. mark doerr university greifswald

Oligo - ε -caprolactone cascade (1) Schmidt, S.; Scherkus, C.; Muschiol, J.; Menyes, U.; Winkler, T.; Hummel, W.; Gröger, H.; Liese, A.; Herz, H.-G.; Bornscheuer, U. T. An Enzyme Cascade Synthesis of ε-Caprolactone and Its Oligomers. Angew. Chem. Int. Ed. 2015, 54 (2), 2784–2787. (2) Dörr, M.; Fibinger, M. P. C.; Last, D.; Schmidt, S.; Santos-Aberturas, J.; Böttcher, D.; Hummel, A.; Vickers, C.; Voss, M.; Bornscheuer, U. T. Fully Automatized High-Throughput Enzyme Library Screening Using a Robotic Platform. Biotechnol. Bioeng. 2016, n/a-n/a. 15 2017-11-09 dr. mark doerr university greifswald

oligo-ε-caprolactone Oligo-ε-caprolactone produced in a biocatalysis reaction starting from 1 M ε-caprolactone using lipase CAL-A in distilled water. After 2 days incubation at 4°C a white sediment was observed that represents the crystallized oligo-ε-CL. Ref.: Schmidt, S.;et al.. . Angew. Chem. Int. Ed. 2015, 54 16 2017-11-09 dr. mark doerr university greifswald

monitoring of bacterial growth 17 2017-11-09 dr. mark doerr university greifswald

18 2017-11-09 dr. mark doerr university greifswald

bacterial growth importance of even growth 19 2017-11-09 dr. mark doerr university greifswald

Towershaker 20 2017-11-09 dr. mark doerr university greifswald

Cytomat 2 contactless switch upgrade

update contactless switches mechanical switches

Javier Geofgrey -Geofg- Yannick Santos S. A. J. Aberturas Waldo Brandson the splitGFP system Santos-Aberturas, J.; Dörr, M.; Waldo, G. S.; Bornscheuer, U. T. In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization. Chemistry & Biology 2015, 22 (10), 1406–1414. PDB: 2B3P

the splitGFP concept 1 11 b -sheets Cabantous, S.; Waldo, G. S. In Vivo and in Vitro Protein Solubility Assays Using Split GFP. Nat Meth 2006, 3, 845–854.

GFP-11-tag Cabantous, S.; Waldo, G. S. In Vivo and in Vitro Protein Solubility Assays Using Split GFP. Nat Meth 2006, 3, 845–854.

S-Aminotransferase Vibrio fmuvialis - GFP-11-taged (cartoon) aminotransferase Vibrio fmuvialis (pdb: 4E3R) Midelfort, K. S.; et al., Protein Engineering (tagging of only one monomer shown) Design and Selection 2013, 26, 25–33.

S-Aminotransferase Vibrio fmuvialis - GFP-11-taged (cartoon) aminotransferase Vibrio fmuvialis (pdb: 4E3R)

S-Aminotransferase Vibrio fmuvialis - GFP-11-taged (cartoon) 530 nm 488 nm

splitGFP for synthetic biology applications ● mulitcolour detection of difgerent protein expression levels ● ● promotor design ● ● Substrate production – reporter functi on Ref. Santos-Aberturas, J.; Dörr, M.; Waldo, G. S.; Bornscheuer, U. T. In-Depth High-Throughput Screening of Protein Engineering Libraries by Split-GFP Direct Crude Cell Extract Data Normalization. Chemistry & Biology 2015, 22 (10), 1406–1414.

microfmuidics : enzyme cascades 30 2017-11-09 dr. mark doerr university greifswald

microfmudics Image: courtesy of Simon Godehard 31 2017-11-09 dr. mark doerr university greifswald

applications of microfmuidics ● in-vitro translation of individual protein variants ● variation of reation conditions by programmatically controled pumps ● deep mutational screening ● coupling of genotype with phenotype ● sorting with double emulsions (water-oil-water) in F luorescence- A ctivated C ell S orting (FACS) ● Sorting on chip 32 2017-11-09 dr. mark doerr university greifswald

acknowledgements Prof. Uwe T. Bornscheuer fjnancial support: DFG and federal state of Mecklenburg-Vorpommern Thermo Laboratory Automation & Cytomat for the invitation and the support in the “contactless switch project” Thank you for your attention ! Any questions ? lara.uni-greifswald.de 33 cartoon: http://xkcd.com/ 2017-11-09 dr. mark doerr university greifswald

Summary ● 34 2017-11-09 dr. mark doerr university greifswald

assays currently possible on the platform acetophenone-types assay pNPA-types assay NADPH assays (BMVO/CHMO) phenol red assay calcein assay retroaldol assay Kemp-eliminase assay longterm stability assays bioassays (Pimaricin, Antifungal, Antibiotics) splitGFP assay

applications of microfmuidics: double emulsion droplet formation Image: courtesy of Simon Godehard 36 2017-11-09 dr. mark doerr university greifswald

FACS Source: wikipedia 37 2017-11-09 dr. mark doerr university greifswald

droplet sorting (1) Gielen, F.; Hours, R.; Emond, S.; Fischlechner, M.; Schell, U.; Hollfelder, F. Ultrahigh-Throughput–directed Enzyme Evolution by Absorbance-Activated Droplet Sorting (AADS). PNAS 2016, 113 (47), E7383–E7389. 38 2017-11-09 dr. mark doerr university greifswald

visions / connections / questions ● new (?) metabolic pathways prediction ● smart design / evolution of experiments ? ● prediction of reaction outcome (product distribution ?) ● prediction of new reactivities ● closing the loop: selection criteria for evolution experiments (?) and manipulation of genetic/chemical sequence/ structure information to feedback on and evolve on a given system (to prevent chemical explosion) 39 2017-11-09 dr. mark doerr university greifswald