Microbiological Methods

2.8.1 Preparation of Chemical Competent Escherichia coli Cells

Many bacterial strains have the natural ability for the admission of external DNA by natural competence mechanisms.[217] In this work, the chemical transformation was used to introduce plasmid DNA into competent E. coli cells.[217, 218] To achieve a high amount of transformants the natural competence was increased by using a standard preparation protocol for E. coli.[217, 218] An

E. coli pre-culture was grown in 4 mL LB media (250 rpm, 16 h, 37°C). One milliliter of pre-culture

was used to inoculate 200 mL LB media in a 1000 mL shake flask. The main culture was grown until OD600 0.4 (250 rpm, 37°C). Subsequent steps were performed on ice or 4°C. Residual LB media was removed by centrifugation (4500 x g, 10 min, 4°C). The obtained cell pellet was resuspended in 15 mL TFB1 solution (30 mM K-Acetate, 50 mM MnCl2, 100 mM RuCl2, 10 mM CaCl2, 15 % glycerol; pH 6.8; sterile filtrated; stored at 4°C) and incubated for 10 min on ice. Resuspended cells were centrifuged and resuspended in 2 mL TFB2 solution (10 mM MOPS, 75 mM CaCl2, 10 mM RuCl2, 15% glycerol; pH 6.8; sterile filtrated; stored at 4°C). TFB1 and TFB2 were sterilized using a sterile 0.2 µM filter. Cells resuspended in TFB2 solution were aliquoted into sterile and pre-cooled 2 mL plastic tubes. Aliquots of 100 µL competent cells were directly frozen in liquid N2 and stored until use at -80°C.

2.8.2 Transformation of Plasmid DNA into Escherichia coli

For transformation of plasmid DNA 100 µL of frozen competent E. coli cells and plasmid DNA were thawed on ice (15 minutes). Commonly, 1 µL containing total 5 ng of isolated and purified plasmid DNA were mixed with 100 µL of chemical competent cells. Cells and plasmid DNA were incubated (15 min; on ice) prior to transformation by heat shock.[217] The transformation was achieved by placing tube with cells and plasmid DNA into a water bath (42°C) for 45 sec. After the heat shock, tubes were placed on ice for five minutes. Subsequently 900 mL of pre-heated (37°C) SOC media (section 2.7.1) were supplemented to the transformation mixture. Cells were recovered for 45 min (250 rpm, 37°C) before plating on LB agar plates with corresponding antibiotics (section 2.7.3). The competence of new generated batches of competent cells was always confirmed by transforming cells with 1 ng pUC19 plasmid DNA.[219] The transformed and recovered cells were plated subsequently in dilutions of 1:10, 1:25, 1:50 and 1:100 (in triplicate) on LB agar plates containing the

33 corresponding antibiotics. Cells were grown overnight at 37°C and counted on the next day for statistical evaluation.[217, 218] The amount of cells and the corresponding dilution factor were used to calculate the competence of the prepared cells with following equation:

Transformation efficiency [cfu µg-]1 = Number of colonies on an agar plate x dilution factor x µg-1 DNA

2.8.3 Preparation of Escherichia coli Cryo-Cultures

Glycerol stocks of bacterial strains were prepared by striking one colony from a plasmid transformation (section 2.8.2) with a sterile inoculation loop on a LB agar plate containing appropriate antibiotics. After incubation (16 h, 37°C), the grown cells were removed from the agar plates using a sterile inoculation loop. Cell mass was resuspended in an autoclaved plastic test tube filled with 1 mL 50 % glycerol (w/w). The cells were resuspended by vortexing the tube thoroughly prior to long time storage at -80°C.

2.8.4 Shake Flask Expression of P450 BM3

Pre-cultures for expression in shake flasks were prepared by inoculating a sterile glass tube containing 3 mL LB media with cells from the glycerol stock using a sterile inoculation loop (section 2.8.3). Cells were grown until reaching stationary phase (250 rpm, 16 h, 37°C). From the pre-culture 1 mL of culture broth was transferred into a sterile Erlenmeyer flask (1000 mL) containing 100 mL TB media, trace elements as well as appropriate antibiotics. P450 BM3 expression was induced by addition of IPTG, ALA and thiamine at optical density of 0.8 at 600 nm wavelength (OD600).[160] Cells were harvested after 16 h of expression by centrifugation (4500 x g, 15 min, 4°C). After discarding remaining media, the pellets containing expressed P450 BM3 were stored until use at -20°C. As a control the expression protocol was performed in the same way with the empty vector plasmid containing no gene for heterologous expression (section 2.6).

2.8.5 Cell Lysis with High-Pressure Homogenizer and Sonicator

Frozen cell pellets from shake flask expressions were thawed on ice and resuspended thoroughly in 10 to 20 mL KPi buffer (50 mM; pH 7.5) until no solid particles were visible. Efficient cell disruption was achieved by a high pressure homogenizer (“French press”) performing three iterative cycles at 1500 bar pressure. Cell debris was removed by centrifugation in an ultra centrifuge (24000 x g, 15 min, 4°C). The supernatant was stored at 4°C until and used for P450 quantification with the CO binding assay (section 2.10.8). Alternatively, smaller cell pellets were lysed by ultrasonication. Therefore the cell pellet was thawed on ice and resuspended in KPi buffer (50 mM, pH 7.5) thoroughly by vortexing. Resuspended cells were placed on ice and disrupted by sonication. Following sonication program was employed for three cycles: 40 % amplitude, 3 cycles à 30 s, 30 s break (cooling). Finally lysed cells were centrifuged (20000 x g, 10 min, 4°C) for entire removal of cell debris and the clear supernatant was used for further experiments.

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2.8.6 Mutant Library Preparation and Expression in Microtiter Plates

Mutant colonies were picked with sterile toothpicks from agar plates into flat bottom 96well MTPs filled with 100 µL of LB media (section 2.7.1) and appropriate antibiotics. Screening for improved variants requires reliable references (positive and negative controls). As negative control on each MTP serves the plasmid without any cloned gene within the multiple cloning site (MCS), the empty vector (EV) (Figure 12 and 13). For a positive control in a screening experiment and for selection of improved variants serves the wild-type (WT) or the best enzyme variant from a previous round of screening. To ensure a high confidence level for selection of improved variants, both controls were placed in triplicate on each MTP (Figure 14).

Figure 14. Scheme for MTP arrangement of generated variants (white colored wells) together with a positive control (e.g. WT; red colored wells: A1; D6 and H12) and negative control (EV; black colored wells; A12, E7 and H1) for reliable screening.

After each well was inoculated with the respective colony from an agar plate the plate was closed with a sterile lid and sealed with tape to prevent evaporation. Cells were grown overnight in a MTP shaker (900 rpm, 37°C, humidity control). To grown cultures 100 µL of 50 % (W/V) sterile glycerol solution were supplemented and the plates stored at -80°C prior to expression and screening for improved variants (Figure 15).

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Figure 15. Preparation and expression process for mutant libraries in MTP format. Library preparation: After chemical transformation clones are picked into a MTP pre-filled with LB media. The plate is incubated for 16 h to grow cells to high density. Finally glycerol is supplemented prior to long time storage at -80°C. Library Expression: Frozen plates were thawed at RT for 15 min and cell mass is replicated into a new MTP filled with LB. After 16 h cultivation at 900 rpm and 37°C, 6 µL from each well were transferred into a deep well MTP filled with TB media and all additives necessary for expression of P450 BM3.[160, 161] Protein expression was performed for 16 h at 900 rpm and 30°C. Residual media was removed by centrifugation and the plates were stored at -20°C until use.

Glycerol stocks of mutant libraries in MTPs were thawed at RT for 15 min. After the plates thawed completely, the cells were replicated with a sterile metal-pin replicator into flat-bottom 96 well MTPs filled with 150 µL sterile LB media and the corresponding antibiotics. Inoculated plates were closed with a sterile MTP lid and sealed with tape to prevent evaporation. Pre-culturing of libraries was achieved in a MTP shaker (900 rpm, 37°C, humidity control) for 16 h. Six µL from each well of the pre-culture were pipetted into 600 µL TB media pre-filled into a 96 deep-well MTP. The TB-media was supplemented with all additives necessary for reliable expression of P450 BM3 (section 2.7.1).[160] Deep well MTPs were closed with a sterile lid and sealed with tape. Expression in deep well MTPs was performed for 20 h in a MTP shaker (900 rpm, 30°C). After expression of mutant libraries the MTPs were centrifuged to remove residual media (3400 x g, 15 min, 4°C). Cell pellets were stored in deep well MTPs for at least 12 h at -20°C until use in screening assays (section 2.10).

2.8.7 Preparation of Microtiter Plates for Screening

Deep well MTPs containing pellets of expressed P450 BM3 monooxygenase were thawed at RT for 15 min. Subsequently 150 µL KPi buffer (50 mM, pH 7.5) were supplemented and the MTP incubated for 5 min at RT. Pellets were resuspended thoroughly by vortexing deep well MTPs until all pellets were entirely resolved. For full lysing of E. coli BL21 lacIQ1 cells additional 150 µL KPi buffer (50 mM, pH 7.5) containing 5 mg mL-1 lysozyme were supplemented to the resuspended pellets.[160] The plates were closed with a lid and incubated for 1 h in a MTP shaker (900 rpm, 37°C). Cell debris, insoluble or solid compounds were removed by centrifugation (3400 x g, 15 min, 4°C). Cell lysates in

36 deep well MTPs were stored at 4°C until final application in activity screening with the respective assays (section 2.10.2 and 2.10.3).