5.2 Media optimization
Firstly, a preliminary screening of various factors was performed using
the Plackett-Burman Design (PBD) method to identify the most influential
variables. Subsequently, the Central Composite Design (CCD) method of
Response Surface Methodology (RSM) was employed to optimize the levels
of these significant variables. All experimental procedures were
conducted in 2 mL volume wells within the Biolector XT Microbioreactor,
each containing 1 mL of the respective culture media. Prior to the
experiments, the media were prepared according to the specific design
points and then inoculated with an overnight seed culture. The
inoculated cells grew for 4 hours in each well, with constant agitation
at 800 rpm, and maintained at 37°C. To facilitate these experiments,
48-well FlowerPlates (mp2-labs, Baesweiler, Germany) was used, with a
working volume of 1000 µL. Throughout the study, real-time monitoring of
growth kinetics was conducted by continuously measuring scattered light
intensities. Additionally, the final insulin density, expressed in grams
per liter, was determined using a UV/Vis spectrophotometer at a
wavelength of 280 nm. To further analyze the results, SDS-PAGE (Sodium
Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) was performed.