High Throughput Screening
In vivo flow cytometry based screening platform
Within HTS division we have developed several in vivo flow cytometry based screening platforms for efficient engineering of protease, glucose oxidase, arginine deiminase, monooxygenase, cellulase, phytases, lipases, and esterases. The main principle of the assay is based on fluorescent labelling of the cells containing active enzyme variants which subsequently can be analyzed and sorted using flow cytometry as depicted in Figure 1. The recent highlight in HTS was a development of in vivo polymer based high throughput screening system Fur Shell for directed evolution and enzyme discovery by providing a technology platform for hydrolases see Publication highlights.Bio VI
Figure 1: Principle of in vivo flow cytometry based screening platform. Mutant library generation and cloning into host cells (1), subsequent expression (2) and fluorogenic reaction catalyzed by expressed enzyme variant (3), analysis and sorting of fluorescently labelled cells containing active enzyme variants using flow cytometer (4), enrichment in population of fluorescently labelled cells (5), cell recovery and activity profile characterization on agar plates and /or MTP (6), and isolation of genes encoding for active enzyme variants (7).Figure adapted from G. Körfer PhD thesis ““Development of a flow cytometer-based in vitro compartmentalization screening platform for directed protein evolution.
In vitro based screening platform
The main principle of in vitro technology platform relies on cell like compartments i.e. double emulsions comprising an inner aqueous phase containing a gene mutant library, a cell-free transcription-translation reaction mixture, and a fluorogenic detection system for activity. The double emulsion compartments mimic the bacterial cell membrane by encapsulating ideally one DNA molecule per compartment and thereby enabling genotype-phenotype linkage. Upon in vitro transcription-translation of the mutant library into enzyme variants, active variants convert a fluorogenic substrate into a fluorescent product thus labelling double emulsion compartment. In vitro compartmentalization IVC technology enables the miniaturization of reaction volumes by production of 1010 reaction compartments per milliliter of reaction with diameters ranging from 0.5–10 μm, thus resulting in a dramatic reduction of consumable costs, workload and assay time. Main advantage of in vitro compartmentalization is its independency from host cells, enabling the production of toxic and membrane bound proteins. Additionally, in vitro based screening system overcomes the challenge of diversity loss in directed evolution experiments due to low transformation efficiency of expression hosts.Bio VI
Figure 2: Principle of in vitro screening platform in double emulsion compartments. Generation of a mutant library using linear DNA (1) and subsequent encapsulation in single water in oil (2) and water in oil in water emulsions (w/o/w). Analysis and sorting of a encapsulated mutant library using flow cytometry (4), Recovery of DNA and PCR amplification (5), cloning and transformation intro expression hots (6), and final characterization of the enriched mutant library in MTP assay (7). Figures adapted from G. Körfer PhD thesis ““Development of a flow cytometer-based in vitro compartmentalization screening platform for directed protein evolution.”
Developed screening technology platforms in MTP and HTS are used within the group for successful directed evolution of proteases, phytase, glucose oxidase, lipases, cellulase, esterases, arginine deiminase, and monooxygenase (Figure 3). Current running projects within HTS division are funded by Federal Ministry of Education and Research, BMBF, and comprise further technology advancements of high throughput screening technology platform.
Additional research areas within HTS division are to expand Fur-shell technology to all kind of hydrolases and oxidases, to understand the principles governing ionic liquids and organic solvents resistance in BSLA and to screen and identify breeding new hydrolases generated by PTRec.
In the division HTS a broad interdisciplinary team is assembled with a competence in polymer chemistry, physical chemistry, molecular biology, and biochemistry. The division HTS harbors as core facility BD influx flow cytometer to enable ultrahigh throughput screenings within directed evolution campaigns.Bio VI