Material and methods

Staining of amyloid protein plaques in mammalian cells using Thioflavin-S is a standard technique. As described before bacterial inclusion bodies display similar protein characteristics like amyloid plaques and Thioflavin-S was already positively applied for comparative quantification of inclusion bodies under varying cultivation conditions. Now, the published protocols were adapted to record the formation of inclusion bodies of an miniproinsulin, interleukin-2, and an alcohol dehydrogenase in E. coli W3110M and RB791.

This approach is separated into three parts:

(1) Test of the available method with two recombinant E. coli strains producing a miniproinsulin, human interleukin-2 and an alcohol dehydrogenase on agar plates.

(2) Monitoring of a test cultivation using E. coli W3110M expressing the miniproinsulin.

(3) At-line monitoring of three recombinant E. coli shake flask cultivations using global fluorescence and flow-cytometry.

106 4.4.3.1. Strain

E. coli K12 W3110M with a mutation triggering the overexpression of the lac inhibitor and its recombinant derivatives W3110M pSW3 (plasmid encoding for recombinant miniproinsulin expressed as inclusion bodies and ampicillin resistance gene) and W3110M pCTUT7-IL2 (plasmid encoding for recombinant human interleukin-2 expressed as inclusion bodies and chloramphenicol resistance gene) were used in almost all experiments. E. coli RB791 is like W3110M and carries the lacI^Q mutation. It was transformed with pADH (plasmid encoding for alcohol dehydrogenase and ampicillin resistance gene). Strain W3110M and plasmid pSW3 were thankfully provided by Sanofi-Aventis Deutschland GmbH. The strain RB791 and the plasmids pCTUT7-IL2 and pADH were obtained from the laboratory strain and plasmid collection. The backbone of pCTUT7-IL2 is part of the plasmid library described in Kraft et al.

(2007).

4.4.3.2. Media

All chemicals mentioned were acquired from either Carl Roth GmbH, Karlsruhe, Germany, or Sigma-Aldrich Chemie GmbH, Munich, Germany, if not otherwise specified. The composition of standard LB medium used for initial precultivations was 10 gL^-1 tryptone, 5 gL^-1 yeast extract, 10 gL^-1 NaCl and appropriate antibiotics (100 µgL^-1 ampicillin, 34 µgL^-1 chloramphenicol). For the first test of Thioflavin-S staining on plates, 15 gL^-1 agar-agar and if needed antibiotics like mentioned before as well as IPTG to a final concentration of 1 mM were added to LB medium before pouring.

The bioreactor cultivation to test monitoring as well as to produce biomass for parameter optimization was done in a 2 L-scale bioreactor. Biomass was harvested as 20 mL aliquots in 50 mL centrifugation tubes, centrifuged at 4°C and 15000 g for 10 min, and stored at -20°C until further use. The used minimal medium (B. Xu, Jahic, and Enfors 1999) in the bioreactor contained 2 gL^-1 Na2SO4, 2.468 gL^-1 (NH4)2SO4, 5 gL^-1, NH4Cl, 14.6 gL^-1 K2HPO4, 3.6 gL^-1 NaH2PO4

x 2H2O, 1 gL^-1 (NH4)2-H-Citrat, 1 mL Antifoam Sigma 204. Per liter medium 2 mL trace elements, 2 mL MgSO4-solution (1.0 M), 2 mL thiamine solution (50 gL^-1), and 1 mL ampicillin solution (100 mgL^-1) were sterile-filtered through an 0.22 µm-membrane filter into the reactor after sterilization. The trace element-solution consisted of 0.5 gL^-1 CaCl2 x 2H2O, 0.18 gL^-1 ZnSO4 x 7H2O, 0.1 gL^-1 MnSO⁴ x H2O, 20.1 gL^-1 Na-EDTA, 16.7 gL^-1 FeCl3 x 6H2O, 0.16 gL^-1 CuSO4 x 5H2O, 0.18 gL^-1 CoCl2 x 6H2O. The starting batch glucose concentration was 5 gL^-1.

107 EnPresso® B (BioSilta Ltd., Cambridge, UK) was used in the final characterization experiments in shake-flasks.

4.4.3.3. Testing Thioflavin-S staining on recombinant model systems

Twenty milliliters of LB medium were inoculated with 50 µL of cryostock and incubated at 37°C for five hours at 200 rpm in a 100 mL Erlenmeyer flask. 200 µL were spread on freshly prepared LB-agar plates containing the appropriate antibiotic and IPTG for recombinant strains and incubated overnight at 37°C. Then the cells were washed from the plates with one mL PBS (8.0 gL^-L NaCl, 0.2 gL^-1 KCl, 1.42 gL^-1 Na2HPO4, 0.27 gL^-1 KH2PO4) and adjusted to an OD600 of 1.

Thioflavin-S staining was done based on the protocol published by Espargaró, Sabate, and Ventura (2012) with the modifications from Aguilera et al. (2016). Cells were washed twice with one mL fresh PBS with centrifugation steps at 5000 g and 4 min. The published 1100 g as centrifuge setting resulted in too soft cell pellets and biomass loss during sample preparation.

After washing, the cell pellet was resuspended in 500 µL Thioflavin-S solution (5 % (w/v) in 12.5 % ethanol) and incubated for 60 minutes at room temperature. After incubation, the stained cell pellet was washed three times with fresh PBS. Finally, the pellet was again resuspended in 1 mL fresh PBS, and 4 * 200 µL were transferred into wells of a UV-transparent multi-well plate (UV-Star® 96-Well Microplates, Greiner Bio-One, Kremsmünster, Austria).

Fluorescence (excitation: 375 nm, emission: 455 nm) and OD600 were measured in a Synergy™

Mx Microplate Reader from BioTek Instruments. The internal detector sensitivity was set to 100.

4.4.3.4. Precultivation for the bioreactor cultivation

Twenty milliliters of sterilized LB medium were inoculated with 50 µL cryostock of the desired strain and incubated for five hours at 37°C and 200 rpm in a 100 mL glass Erlenmeyer flask.

For the second precultivation, 100 mL mineral salt medium were inoculated with one mL LB-preculture standardized to OD600 1 in a 500 mL Erlenmeyer flask and cultivated like before. As the OD600 reached 0.3, the total broth volume was taken and transferred into the bioreactor for a final OD600 of 0.015.

108 4.4.3.5. Cultivation conditions for the bioreactor cultivation

The bioreactor cultivation was performed as pulsed-fed fed-batch experiments using E. coli W3110M pSW3 in a KLF 2000 with a total volume of 3.7 L from Bioengineering AG (Wald, CH).

2 L of the earlier defined mineral salt medium in the reactor vessel were inoculated with 5 % (v/v) preculture broth. After the batch phase feeding was started at substrate depletion. The feeding solution contained 440 gL^-1 dextrose solved in fresh cultivation medium. Changes were a four times increased trace element concentration to avoid limitations at high cell densities as well as no MgSO4, antifoam or ampicillin. Extra 4 mL 1.0 M MgSO4-solution were added every OD600 20. The feed inlet was connected to the top gas phase of the STR.

The feeding phase was divided into two parts. The first part was performed as exponential feeding phase with an initial feeding rate F0 = 0.0125 Lh^-1 and an exponential growth of µ = 0.3. After three hours of feeding, recombinant protein synthesis was induced by addition of IPTG to a final concentration of 1 mM. From now on, the feeding rate was no longer increased.

In this pulsed-fed cultivation, a nutrient oscillation cycle of 10 minutes was used feeding the whole glucose of one cycle in the first minute followed by a 9-min recovery. After 6 hours of additional feeding, pulsed-feeding was stopped and switched to constant feeding with a rate of F = 0.016 Lh^-1. The cultivation temperature was regulated at 35°C during the whole process.

The aeration rate was set to 0.5 vvm in the beginning and raised to 1.0 vvm after feed start.

The pH was controlled at pH = 7.0 with 25 % NH4+ solution.

4.4.3.6. Analytical Methods

Optical density measurements monitored cell growth at a wavelength of 600 nm (OD600) (Novaspec III by Amersham Biosciences, Amersham, UK) in addition to dried cell weight (DCW) determination. To measure DCW 2 mL of cell suspension were transferred into a dried, pre-weighted two mL microcentrifugation tube. After centrifugation for 10 min at 21,500g, the supernatant was discarded, and the cell was resuspended in 1 mL 0.9 % (w/v) NaCl solution.

Following a repeated centrifugation step the tube was dried 75°C for 24 h.

For the weight measurements of inclusion bodies protein fractions, samples were purified using the BugBuster® Protein Extraction Reagent (Merck, Darmstadt, Germany).

Data plots were created with Qtiplot (Qtiplot.com), MODDE 10 (MKS Data Analytics Solution, Malmö, Sweden) and Excel 2016 (Microsoft, Redmond, USA).

109 4.4.3.7. Shake-flask cultivations using EnPresso® B

Twenty milliliters of fresh LB medium were prepared and mixed with 50 µL of E. coli cryostock and incubated for eight hours at 200 rpm and 37°C. For the main cultivation, 25 mL Enpresso®

B medium was completed with each 25 µL ampicillin or chloramphenicol stock and BioSilta Reagent A along with 250 µL LB culture and cultivated for around 14 h at 37°C and 250 rpm in a PreSens SFR flask mounted on the SFR platform. Using PreSens flasks enabled on-line monitoring of DOT and pH levels. Then, protein expression was induced in recombinant cultures by addition of IPTG to a final concentration of 1 mM. In addition, the feeding rate was doubled with extra 25 µL Reagent A per flask.

4.4.3.8. SDS-PAGE

Insoluble protein expression was checked via SDS-PAGE analysis. Cell samples were collected and normalized to an OD600 18 and treated with the BugBuster® Protein Extraction Reagent (Merck, Darmstadt, Germany). Each 10µL of the soluble and insoluble protein fractions were mixed with 20µL demineralized water and 30µL of 2x loading buffer (100 mM Tris-Cl (pH 6.8), 20% glycerine 4 % SDS, 0.2 % bromphenol blue, 200 mM DTT). Mixed samples were incubated for 5 min at 95°C. After cooling down to room temperature, polyacrylamide gels (5 % stacking, 12 % separation) were loaded either with 10 µL of sample, 5 µL of Roti®-Mark TRICOLOR size marker or 10 µL of 2x loading buffer (empty pockets). The electrophoresis was run at 64 V for 30 min followed by around 90 min at 120 V.

Afterwards, the gels were washed to remove residual SDS and stained with Coomassie solution (60 – 80 g Coomassie® Brilliantblue G250 solved in 1L demineralized water and stirred for 2 – 3 hours followed by addition of 35 mM HCl) overnight. After 18 h the Coomassie solution was discarded and the gels were washed in demineralized water to remove the remaining dye and finally photographed (for a detailed protocol see SOP 9.3).

4.4.3.9. Fluorescence Microscopy

Microscopic observations were done with a DMI6000 B (Leica, Wetzlar, Germany) inverse microscope equipped with a 63x/NA 1.40 oil immersion objective. Thioflavin-S fluorescence was recorded using a GFP filter exciting from a range from 450 – 490 nm. Emission was detected in a range from 500 – 550 nm. Digital images were recorded with Leica LAS X. Stained cell samples were prepared as described in 4.4.3.3.

110 4.4.3.10. Flow-Cytometry

Thioflavin-S staining for flow cytometry analysis was done as described in 4.4.3.3. Flow cytometry measurements were performed using a Miltenyi MacsQuant flow cytometer.

Before and after staining cells were at first analyzed by forward (FSC) and side scatter (SCC) signals recorded at 561/10 nm, and then characterized for Thioflavin-S fluorescence by exciting at 405 nm and registering the emission at 450/50 nm.