The wblI coding sequence was amplified from the genomic DNA of S. lividans TK24 by PCR using primers ExpWblI-NdeI and ExpWblI-XhoI. The resulting DNA fragments were cloned into pET22b(+) cut by NdeI and XhoI, and the resultant plasmid transformed into E. coli BL21, a suitable host for protein expression. The resulting transformants were cultivated in LB medium supplemented with 50 μg/ml ampicillin at 37°C until OD600 reached 0.6. IPTG was then added at a final concentration of 1 mM, and incubation was pursued for 6 extra hours. The cells were collected by centrifugation at 12,000 g for 10 min. Then the cell pellet was resuspended in lysis buffer (50 mM Na2HPO4, pH 8.0, 500 mM NaCl) and sonicated (Hielscher Ultrasonics UP400S, 0.5 cycle and 20% amplitude) on ice until reaching complete homogeneity. After centrifugation at 14,000 rpm for 20 min, the cell extract was saved and passed through an Ni-NTA column (Cat. No. 30210; Qiagen) on a Biologic LP apparatus (Bio-Rad). The six-histidine-tagged WblI (His6-WblI) was purified to near homogeneity according to the manufacturer’s instructions.
Ultrasonics up400s
Manufactured by Hielscher
The Ultrasonics UP400S is a high-performance ultrasonic processor designed for laboratory applications. It features a powerful 400-watt ultrasonic generator and a titanium sonotrode (ultrasonic probe) that can be used for tasks such as cell disruption, homogenization, and dispersion of samples. The device operates at a frequency of 24 kHz and provides adjustable amplitude control.
Automatically generated - may contain errors
2 protocols using ultrasonics up400s
1
Overexpression and Purification of WblI Protein
2
Cell Staining and Flow Cytometry
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Cells were harvested, washed with PBS and stained for 10 min in the dark with 1mg/ml FITC (Sigma-Aldrich) in carbonate buffer (0.1M, pH 9.5). Cells were washed 3 times with PBS and visualized by fluorescence microscopy as described below.
Flow cytometry GFP and mCherry-expressing cells were harvested and fixed with 4% paraformaldehyde for 15 min at room temperature. Paraformaldehyde was quenched with glycine 0.5M for 15 min at room temperature. Cells were washed 2 times with PBS, resuspended in PBS and sonicated for 10 seconds at 30% amplitude (Hielscher Ultrasonics UP 400S). Flow cytometry was performed using an LSR II Flow Cytometer (BD Biosciences). Data analysis and plotting was performed in R.
Flow cytometry GFP and mCherry-expressing cells were harvested and fixed with 4% paraformaldehyde for 15 min at room temperature. Paraformaldehyde was quenched with glycine 0.5M for 15 min at room temperature. Cells were washed 2 times with PBS, resuspended in PBS and sonicated for 10 seconds at 30% amplitude (Hielscher Ultrasonics UP 400S). Flow cytometry was performed using an LSR II Flow Cytometer (BD Biosciences). Data analysis and plotting was performed in R.
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