Cellulolytic RoboLector - towards an automated high-throughput screening platform for recombinant cellulase expression

被引:25
作者
Muehlmann, Martina [1 ]
Kunze, Martin [1 ]
Ribeiro, Joaquim [1 ]
Geinitz, Bertram [1 ]
Lehmann, Christian [2 ]
Schwaneberg, Ulrich [2 ]
Commandeur, Ulrich [3 ]
Buechs, Jochen [1 ]
机构
[1] Rhein Westfal TH Aachen, AVT Chair Biochem Engn, Worringerweg 1, D-52074 Aachen, Germany
[2] Rhein Westfal TH Aachen, Chair Biotechnol, Worringerweg 1, D-52074 Aachen, Germany
[3] Rhein Westfal TH Aachen, Chair Mol Biotechnol, Worringerweg 1, D-52074 Aachen, Germany
来源
JOURNAL OF BIOLOGICAL ENGINEERING | 2017年 / 11卷
关键词
High-throughput screening; Automation; On-line monitoring; Cellulase; Microtiter plate; ESCHERICHIA-COLI; PROTEIN EXPRESSION; MICROTITER PLATE; SHAKE-FLASK; FLUORESCENCE; CULTIVATION; BIOMASS; GROWTH; PURIFICATION; FERMENTATION;
D O I
10.1186/s13036-016-0043-2
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Background: Cellulases are key player in the hydrolyzation of cellulose. Unfortunately, this reaction is slow and a bottleneck in the process chain from biomass to intermediates and biofuels due to low activities of the enzymes. To overcome this draw back, a lot of effort is put into the area of protein engineering, to modify these enzymes by directed evolution or rational design. Huge clone libraries are constructed and have to be screened for improved variants. High-throughput screening is the method of choice to tackle this experimental effort, but up to now only a few process steps are adapted to automated platforms and little attention has been turned to the reproducibility of clone rankings. Results: In this study, an extended robotic platform is presented to conduct automated high-throughput screenings of clone libraries including preculture synchronization and biomass specific induction. Automated upstream, downstream and analytical process steps are described and evaluated using E. coli and K. lactis as model organisms. Conventional protocols for media preparation, cell lysis, Azo-CMC assay and PAHBAH assay are successfully adapted to automatable high-throughput protocols. Finally, a recombinant E. coli celA2 clone library was screened and a reliable clone ranking could be realized. Conclusion: The RoboLector device is a suitable platform to perform all process steps of an automated high-throughput clone library screening for improved cellulases. On-line biomass growth measurement controlling liquid handling actions enables fair comparison of clone variants.
引用
收藏
页数:18
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