Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

The overall goal of this iterative workflow combining Kringing based experimental design and microbioreactor technology for sufficient cultivation throughput is to describe a generic approach for tailor made design of microbial cultivation media. This method can help answer key questions in the field of microbial bioprocess development, such as the optimization of cultivation media, bioprocess conditions, as well as strain phenotyping. We first had the idea for this method when we asked ourselves if cultivation media optimized for small metabolite production are also best suited for heterologous protein production.

The main advantage of this technique is that it speeds up overall development times by smart combination of experiment and modeling. Demonstrating this procedure will be Roman Jansen, a PhD student from my laboratory. Begin this procedure with study conception and definition of methods as detailed in the text protocol.

Sterilize deep well plates for stock solution storage on a work table by wiping the plates with 70 percent ethanol and subsequently drying them in a laminar flow hood. Start the seed culture by inoculating 50 milliliters of BHI medium containing 25 milligrams per liter of kanamycin with one aliquot from the working cell bank. Place all necessary labware on the robotic work table and pour stock solutions in the corresponding labware.

Then, start the robotic work flow for media preparation so that the last step, which is the inoculation, is reached in time for the start of seed culture. Sample the seed culture after approximately two hours to monitor the growth by measuring the optical density at 600 nanometers or O-D-600. Continue to monitor the growth at each hour.

After approximately five hours, the culture reaches an O-D-600 between three and four, and is used to inoculate the main cultures. Place the seed culture on the liquid handler work table and continue the media preparation protocol. Seal the cultivation microtiter plates after inoculation.

Next, place the sealed cultivation M-T-P in the Biolector device. Then, start the predefined cultivation protocol. Dispose of the remaining stock solutions according to biosafety regulations, if necessary.

Then, seed the culture from the robotic work table. Clean the reuseable labware before starting the liquid handler decontamination protocol. Stop the main culture after a 17 hour run time and process the data as described in the text protocol.

To quantify the G-F-P titer, first transfer the cell suspensions from all cultivation wells into prelabled reaction tubes. Then, centrifuge the cell suspensions for 10 minutes at maximum speed using a bench top centrifuge to obtain the cell free supernatant. Next, transfer 200 microliters of each cell free supernatant into a black 96 well M-T-P with a transparent bottom.

Read the G-F-P specific fluorescence at 488 nanometers for excitation and 520 nanometers for emission wavelength using an appropriate microplate reader. After measuring the G-F-P fluorescence, the microplate can be directly used for determination of protein content of the cell free supernatants with standard protocols such as a Bradford or B-C-A assay. Finally, proceed to data analysis as described in the text protocol.

The demonstrated procedure constitutes one round of experiments, the results are used for designing further rounds with maximal expected improvement of the G-F-P signal. Reference experiments are placed in each round for analyzing data variability and detecting experimental outliers. This contour plot gives an overview of the optimization procedure.

The analysis of the collected data set through iteration three revealed a limitation of the positive effect of magnesium, identifying an optimal concentration range. It was therefore decided to further expand the concentration range only for calcium in iteration four. The procedure was repeated twice in iteration five and six until a saturation of the G-F-P signal was found.

This saturation is explained by precipitation of calcium salt for the applied concentrations of calcium, which are not available to the cell. The seventh iteration was used for exploring the boundaries in more detail. The optimal concentration ranges of media components for the saturated G-F-P signal can be reliably identified with a statistical Z-test.

It shows the identified plateau as determined and visualized using the Kringing kit tool box. The Kringing kit toolbox is freely available and comes with a detailed tutorial that explains how to use its features. The generic nature of the presented protocol allows various adaptions, such as studying other microbial expression hosts or optimization of other properties of the target protein, like glycosylation pattern or disulfide bonds.

The integration of an embrion system increases experimental throughput, which enables great savings in time When replacing fully controllable and influented bioreactors with amniar systems, scalability of results must be considered. Mathematical modeling and design of experiments helps to maximize the information content of measurement data with respect to the studied objective.