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PhD

Jascha Rolf

Telefon
(+49)231 755-5475

Adresse

Raum G1 6.20

Project

Abstract:
For bioprocess development the screening of suitable biocatalysts is essential and a complex task [1]. Therefore, the aim of my study is the development of a high-throughput screening system for biocatalysts. The rate limiting step in the generation of biocatalyst libraries is typically the heterologous expression and protein purification. Hence, utilizing cell-free protein synthesis (CFPS) in an automatized setup can circumvent this bottleneck and increase the efficiency of bioprocess development.

Description:
Cell-free systems are used for protein synthesis since decades with a wide range of applications and bear several advantages compared to heterologous expression. Proteins can be produced from DNA templates within few hours with concentrations at a milligram per milliliter scale [2]. The CFPS mix consists of three parts:

  • A cell-free extract, which contains all necessary constituents for the coupled transcription and translation machinery, such as ribosomes, aminoacyl‑tRNA‑synthetases, and translation factors for initiation, elongation, and product release.
  • The DNA template with the gene of interest.
  • An energy mix with the building blocks for mRNA and the protein synthesis and additional cofactors and supplements.

The rational improvement of supplemental components results in systems, which permit novel CFPS applications beyond pure research interests. Hence, the integration of CFPS in platforms for high‑throughput enzyme screening and analysis can serve as a powerful and versatile tool for the fast discovery of new candidates or improved biocatalyst variants.

in vitro expression
Figure 1: The principle of cell-free protein synthesis. The cell-free extract contains the machinery for the coupled transcription and translation reaction; the DNA template consists of regulatory sequences and encodes the target protein; the energy mix contains building blocks for mRNA and protein synthesis, as well as components for energy regeneration and supplemental substances.


[1] Lütz, S., Giver, L., & Lalonde, J. (2008). Engineered enzymes for chemical production. Biotechnology and Bioengineering, 101(4), 647–653. doi:10.1002/bit.22077
[2] Caschera, F., & Noireaux, V. (2014). Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie, 99(1), 162–168. doi:10.1016/j.biochi.2013.11.025



Publikationen

Paper

  • Rolf J., Rosenthal K., and Lütz S. (2019)
    Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development
    Catalysts. 2019; 9(2):190

Poster

  • Rolf J, Mattijs JK, and Lütz S (2018)
    Biotechnological Monoterpene Production in Escherichia coli
    ProcessNet-Jahrestagung und 33. DECHEMA-Jahrestagung der Biotechnologen, 10. - 13. September 2018, Aachen, Germany