3 METHODS
3.2 Microbiological methods
3.2.1 Cultivation and storage of E. coli strains
All E. coli strains used were cultivated at 37 °C while shaking at 140 rpm (1 liter cultures) or 220 rpm (5, 50 and 250 ml cultures), respectively. For cultivation LB-medium was used, unless otherwise stated. For plasmid-harboring strains the medium was supplemented with the corresponding antibiotics (150 µg/ml ampicillin, 30 µg/ml chloramphenicol, 75 µg/ml kanamycin, 12.5 µg/ml tetracycline) using a filter-sterilized, thousand fold concentrated stock solution. To obtain single colonies, the cell suspension was plated on agar plates containing the adequate antibiotics, and incubated overnight at 37 °C. For temporary storage the plates or suspensions were sealed and stored at 4 °C. For long-term storage, glycerol cultures were made and stored at -80 °C. For this purpose an aliquot of an overnight culture was mixed in a 1:1 ratio with 87% glycerol, and stored in a sterile screw cap reaction vessel.
3.2.2 Preparation of chemically competent E. coli cells (Inoue et al., 1990)
For preparation of chemically competent E. coli cells, 500 ml SOB medium was inoculated with the respective overnight culture to an OD600 of 0.1, and cultured at 37 °C and 220 rpm until an OD600 of 0.6 was reached. The culture was incubated on
ice for 15 min, transferred into 50 ml tubes, and cells were harvested by centrifugation (EPPENDORF Centrifuge 5810R, 4000 rpm, 10 min, 4 °C). The cell pellet was resuspended in 100 ml ice-cold TFB I buffer, centrifuged a second time under the same conditions as stated above. The resulting pellet was resuspended in 10 ml ice-cold TFB II-buffer. Immediately after resuspension, 100 µl aliquots of the cell suspension were transferred to Eppendorf reaction vessels on ice and stored at -80 °C.
3.2.3 Transformation of chemically competent E. coli cells
For transformation of chemically competent cells a 100 µl aliquot was thawed on ice, and about 100 ng plasmid DNA (maximum volume: 20 µl) was added. Following incubation on ice for 5 min, cells were heat-shocked for 45 s at 42 °C, and incubated again on ice for 5 min. A volume of 900 µl LB-medium was added, and cells were incubated for 1 h at 37 °C in a shaker at 220 rpm to develop antibiotic resistance.
Finally, adequate dilutions of the cell suspension were plated on LB agar plates containing the appropriate antibiotics for selection.
3.2.4 Preparation of electro-competent E. coli cells (Dower et al., 1988) For preparation of electro-competent E. coli cells, 50 ml or 200 ml SOB-medium were inoculated with the respective overnight culture to an OD600 of 0.1, and incubated at 37 °C and 220 rpm until an OD600 of 0.6 was reached. The culture was incubated on ice for 30 min, transferred to 50 ml tubes, and cells were harvested by centrifugation (EPPENDORF Centrifuge 5810R, 4000 rpm, 10 min, 4 °C). The pellet was resuspended in 50 ml ice-cold, sterile water and incubated on ice for another 15 min. Following centrifugation the pellet was resuspended in 20 ml ice-cold, sterile water and incubated again on ice for 15 min. The cells were pelleted by centrifugation and resuspended in 10 ml ice-cold, sterile water. After the last incubation and centrifugation step the supernatant was discarded, and the cells were incubated on ice. The cell pellet was resuspended in a minimum of 1 ml sterile water, and 100 µl aliquots were transferred to Eppendorf reaction vessels. Aliquots of electro-competent cells were directly used for transformation or stored at -80 °C in 10% glycerol.
3.2.5 Preparation of DNA for electroporation
For electroporation the salt content of the DNA solution should be as low as possible, to avoid a short-circuit during the electric pulse. Therefore, the DNA which is generally dissolved in a saline buffer (e.g. ligation mixture) has to be dialyzed prior to transformation. For this purpose 10-20 µl of the DNA solution was placed for at least 1 h onto a nitrocellulose filter (MILLIPORE), which was then put onto the surface of MILLIPORE-water in a small petri dish.
3.2.6 Transformation of electro-competent E. coli cells
For electroporation a maximum of 20 µl salt-free DNA-solution was added to 100 µl electro-competent cells. After 5 min incubation on ice the transformation mixture was transferred into a pre-cooled electroporation cuvette (gap length 2 mm).
Following the electrical pulse (2500 V, 25 µF, 200 Ω) in the electroporator (time constant between 4.0 and 6.0 ms in case of successful electroporation) the cell suspension was immediately supplemented with 1.5 ml SOC medium, and incubated for 1 h at 37 °C in a shaker. Aliquots were plated on selective agar plates, and incubated overnight at 37 °C.
3.2.7 Determination of transformation efficiency
For determination of the transformation efficiency (TE) one aliquot of competent cells was transformed with 100 ng plasmid DNA, as described above (3.2.3 & 3.2.6).
The cells were plated on selective agar using various dilutions (undiluted, 1:10, 1:100, 1:1000 etc.). The transformation efficiency was calculated as follows:
!! = !!"#"$%&'∙ !
!!!"
Equation 1: Determination of the transformation efficiency.
TE transformation efficiency (colonies per µg DNA) ncolonies number of colonies
f dilution factor
mDNA applied DNA amount [µg]
3.2.8 In vitro activity screening
In vitro activity screening is a fast and reliable method used to assay enzyme activities of lysed colony replicates on filter paper (filter paper activity pre-screen) or
in crude extracts (96-well block activity screen). Upon incubation with substrate, OP hydrolysis is detected as a colorimetric signal, which is based on the release of the product p-nitrophenol under basic conditions at 400 nm. An overview of the in vitro activity screening process is illustrated in Figure 9.
Figure 9: Overview of the in vitro activity screening procedure.
Filter paper based and 96-well block activity screening.
In vitro activity screening was used to compare activities of Dr0930 variants and to screen the pTNA-dr0930 and pET24a-dr0930 gene libraries for variants with increased OPH activity. The 96-well block activity screen enabled to analyze several hundred colonies per day.
If not state otherwise, chemically competent E. coli BL21-CodonPlus(DE3) cells were transformed with the pTNA-dr0930 gene library, incubated for 1 h at 37 °C, plated on LB agar plates containing the appropriate antibiotics, and incubated overnight.
In case the DEAE-filter paper activity pre-screen was performed to discriminate active and inactive colonies, cells were plated on large square LB agar plates (245 × 245 × 18, BioDish XL, BD). Colony replicates were generated on Whatman DEAE cellulose filter paper (grade DE81) and the master plate was incubated for additional 6 h at 37 °C. DEAE-filter paper limited the diffusion of p-nitrophenol due to electrostatic interaction of the product with the diethylaminoethyl (DEAE) tertiary amine functional group of the anion exchange cellulose. Colony replicates on the DEAE-filter paper were lysed by incubation with a second Whatman filter paper (standard Whatman cellulose filter, Grade 1) soaked with 1 x BugBuster® protein extraction reagent (MERCK) in 50 mM HEPES pH 8.5, 100 µM CoCl2 and incubated for 15 min. The activity was assayed by subsequent incubating of the replicate filter paper with Whatman filter paper soaked with 0.5 mM ethyl-paraoxon in 50 mM CHES pH 9.0. Active colonies turned yellow upon substrate hydrolysis and were identified
visually or by digital imaging (using a band pass filter of 406.5-8.1 nm). Positive colonies were picked from the master plate after overnight incubation and restreaked to obtain single colonies for subsequent 96-well block activity screening.
Single colonies were used to inoculate 96-well blocks (EPPENDORF, deep-well blocks 96/2000) containing 0.75 ml Super Broth medium supplemented with the appropriate antibiotics and 0.5-1.0 mM CoCl2, unless otherwise stated. The deep-well blocks were covered with a gas-permeable, self-adhesive sealing tape (QIAGEN, air-pore sheets) and incubated overnight at 37 °C, upon shaking at 240 rpm. Each 96-deep-well block contained a set of controls in triplicates, unless otherwise stated. If applicable, an aliquot of the master plate overnight culture was mixed with 65% (v/v) glycerol, 20 mM Tris-HCl pH 5.6, 100 mM MgSO4 in a 1:1 ratio and stored at -80 °C. The cells were lysed by incubation with 1 x BugBuster in 50 mM HEPES pH 8.5, 100 µM CoCl2 and incubated for 30 minutes at RT. To this end, the 1 x BugBuster solution was either directly added to the 96-deep-well block cultures or to an aliquot that was transferred to a new microtiter plate. Subsequently, an aliquot of the crude lysate was used to assay activities for OP substrates 1-7 (p-NP analogues of VX, GB, VR, GD, and GF; MPXN and EPXN; 4.2.2) in 50 mM CHES pH 9.0 using a new microtiter plate. Depending on the OP compound and the stringency of the screen, substrate concentrations of 0.15-0.5 mM were used. If applicable, the crude lysate was diluted in a 1:5 or 1:10 ratio in 50 mM HEPES pH 8.5, 100 µM CoCl2 prior to use. For substrates 4 (GD p-NP) and 5 (GF p-NP) a final concentration of 10% methanol was added to the assay set-up. The release of p-nitrophenol was spectrophotometrically monitored in constant time intervals at 400 nm and 30 °C, using an absorbance plate reader (TECAN, Infinite M200 Pro or MOLECULAR DEVICES, SpectraMax-340 plate reader). Plasmids of the most active colonies were extracted and analyzed by sequencing. In combination with the DEAE filter paper activity pre-screen, the method enables for high-throughput analysis of several thousand colonies per day.