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

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

Mar 20, 2024 121 likes •516 views

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

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  • dr. mark doerr university greifswald

Thermo Workshop Mannheim 2017-11-09/10

On the way to Synthetic Biology:

Enzyme Cascade Optimisation at the University of Greifswald Robotic Platform LARA

including applications of the Cytomat 2 ToS

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  • ur robotic platform

Laboratory Automation Robotic Assistant

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colony picking robot(MD QPix420)

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robotic liquid dispenser (Thermo multidrop)

f a a a s t !

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4 robotic incubators (cytomats 2)

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robotic centrifuge (Hettich Rotanta 460 R)

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liquid handling robot(Agilent Bravo)

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UV/fmuorescence reader units for robots

barcode reader Thermo Varioskan BMG Omega Fluorostar

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central robotic arm (Thermo/Fanuc F5)

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making robot experiments

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Our robotic process

growth (MTP)

OD t

induction centrifugation/ harvesting lysis

AU t

assay data analysis/ statistics

R

2 n n 3 10 n

4 difgerent enzyme classes with difgerent production organisms

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what do we screen ?

Current projects cover:

  • New and altered substrate specifjcity of amino transferases
  • altered enantio-selectivity
  • improvement of enzyme stability (BMVOs)
  • improvement of enzyme solubility (pig liver esterase)
  • de-novo enzyme studies (AlleyCat)
  • in-vitro-expression of enzymes (PURE system)
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synthetic biology applications: enzyme cascades

Sandy Schmidt (TU Graz)

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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.
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  • ligo-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

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monitoring of bacterial growth

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bacterial growth

importance of even growth

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Towershaker

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Cytomat 2 contactless switch upgrade

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update

mechanical switches contactless switches

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the splitGFP system

PDB: 2B3P Javier Santos Aberturas Geofgrey -Geofg- S. Waldo Yannick

  • A. J.

Brandson

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.

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the splitGFP concept 1

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

11 b-sheets

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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.

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S-Aminotransferase Vibrio fmuvialis

  • GFP-11-taged (cartoon)

aminotransferase Vibrio fmuvialis (pdb:4E3R)

Midelfort, K. S.; et al., Protein Engineering Design and Selection 2013, 26, 25–33.

(tagging of only one monomer shown)

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aminotransferase Vibrio fmuvialis (pdb:4E3R)

S-Aminotransferase Vibrio fmuvialis

  • GFP-11-taged (cartoon)
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488 nm 530 nm

S-Aminotransferase Vibrio fmuvialis

  • GFP-11-taged (cartoon)
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splitGFP for synthetic biology applications

  • mulitcolour detection of difgerent protein expression levels
  • promotor design
  • Substrate production – reporter function
  • 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.

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microfmuidics : enzyme cascades

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microfmudics

Image: courtesy of Simon Godehard

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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

Fluorescence-Activated Cell Sorting (FACS)

  • Sorting on chip
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Thank you for your attention !

Any questions ? lara.uni-greifswald.de

cartoon: http://xkcd.com/

acknowledgements fjnancial support: DFG and federal state of Mecklenburg-Vorpommern Thermo Laboratory Automation & Cytomat for the invitation and the support in the “contactless switch project”

  • Prof. Uwe T. Bornscheuer
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Summary

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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

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applications of microfmuidics: double emulsion droplet formation

Image: courtesy of Simon Godehard

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FACS

Source: wikipedia

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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.

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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)