Robotic high-throughput screening for the optimization and prediction of recombinant protein expression in microscale

  • Automatisiertes Hochdurchsatzscreening zur Optimierung und Vorhersage der rekombinanten Proteinexpression im Mikromaßstab

Mühlmann, Martina Julia; Büchs, Jochen (Thesis advisor); Kremling, Andreas (Thesis advisor)

Aachen (2018, 2019)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2018


The aim of this work was to develop methods for a reproducible clone screening as well as methods to increase and predict recombinant protein production in Escherichia coli. Automated high-throughput screenings (HTS) with combinable modules from upstream and downstream processes were realized. It was screened for optimized strains as well as for optimized cultivation and induction conditions. Therefore, a robotic platform (RoboLector), which is equipped with a liquid handling system and a BioLector, was applied. Standard procedures for manual media preparation, cell lysis, protein quantification, enzyme activity detection and sampling have been transferred to automatable protocols. Many parameters affect the recombinant protein expression. In this work, temperature, inducer concentration, and induction time were investigated in more detail due to the lack of broad studies. With the help of the RoboLector, induction profiling was carried out at temperatures of28°C - 37°C in 48-well Flowerplates. Data were additionally compared with shake flask experiments in the RAMOS (Respiration Activity Monitoring System) device. Both, the biomass measurements and the oxygen transfer rate (OTR) data, indicated a stronger metabolic burden from induction at high temperatures than at low temperatures. In addition, even low inducer concentrations (0.5 - 0.1 mM IPTG) were sufficient to achieve high product formation. OTR data provide a lot of information about the metabolic state of a culture. So far, the measurement of OTR in microtiter plates is not possible in commercially available devices. Therefore, the recognized correlations between OTR and biomass-based scattered light were examined in more detail in the context of this work. The first derivative of the scattered light course revealed to be proportional to the OTR trend for E. coli. Thus, the scattered light data can serve for the interpretation of physiological states in many cases. The nictotinamide adeninedinucleotide (NADH) fluorescence of the cells could be identified as an additional signal for metabolic burden. Moreover, two different approaches to predict recombinant protein formation have been developed.