Cytation plate reader

Pseudotyped lentiviral vectors were produced as previously described^{32 (link)}. Briefly, HEK293T cells were co-transfected in a 2:1 ration with the pNL4-3-inGluc vector and the spike plasmid of interest using polyethyleneimine transfection (Transporter 5 Transfection Reagent, Polysciences) to produce pseudotyped lentiviral particles. The lentivirus was collected by taking the media of the transfected cells 48 and 72 hours post-transfection. Relative infectivity of the lentivirus was then assessed in both HEK293T-ACE2 and CaLu-3 cells. Gaussia luciferase activity measured at 72 hours post infection for HEK293T and 72 hours for CaLu-3 were used to determine relative infectivity. Gaussia luciferase activity was determined by taking equal volumes of infected cell media and Gaussia luciferase substrate (0.1 M Tris pH 7.4, 0.3 M sodium ascorbate, 10 μM coelenterazine) and combining for an immediate luminescence signal detected by a BioTek Cytation plate reader.

For primary screening of the synthetic promoter libraries, B. subtilis cells carrying the plasmids were cultivated in 96-well plates filled with LB medium for 24 h. Cell density (OD₆₀₀) and fluorescence intensity (excitation wavelength 490 nm, emission wavelength 530 nm, gain 70) were measured with BioTek Cytation Plate Reader. The culture of B. subtilis strain carrying the pHT01 vector was applied to subtract background fluorescence signal. During the second round of screening, 26 strong promoters that did not impair cell growth were re-assayed in shake flask cultures. Sampling was performed every 4 h, and cells were washed with 20 mM phosphate buffered saline (pH 7.0, PBS). After appropriate dilution, cell density and fluorescence intensity were measured as described above.
To test the response of synthetic promoters to stresses including low pH and high salinity, cells were pre-cultivated at normal condition (LB medium, 37 °C, pH 7.0) for 6 h. Afterwards, the cultures were shifted to the designated conditions by supplementing 20 mM final concentration of sodium citrate buffer (pH 4.5) or 0.5 M final concentration of NaCl. Samples were taken every 2 h. Cells were washed with PBS, and the cell density (OD₆₀₀) and fluorescence intensity were measured as described above.

Pseudotyped virus was produced as previously described.^{4 (link)} In short, HEK293T cells were co-transfected with the pNL4-3-inGLuc vector and spike of interest in a 2:1 ratio using a polyethyleneimine transfection (Transporter 5 Transfection Reagent, Polysciences). Pseudovirus was collected from the media of producer cells 48- and 72- hours post transfection and used to infect HEK293T-ACE2 or CaLu-3 cells. Luminescence derived from secreted luciferase was used as a readout for infectivity and captured by a BioTek Cytation plate reader.

Avidin was immobilized onto 1 and 2 layer pCB using the same procedure as bevacizumab-647. Following immobilization, surfaces were rinsed 6 times with 0.05% TWEEN 20 in PBS (PBS-T). Avidin functionalized wafers and pCB coated wafers without avidin were incubated in 100 μL of 4 μg mL⁻¹ biotin-fluorescein in PBS for 2 h in the dark under agitation (orbital shaker at 100 RPM). The biotin-fluorescein solution was removed and surfaces were rinsed 6 times with PBS-T. Fluorescence of each well was measured with a Biotek Cytation plate reader and surface fluorescence was imaged by fluorescent microscopy. Surface fluorescence values were converted to ng cm⁻² by comparison with a calibration curve of dropcast biotin-fluorescein of known total mass onto wafers and imaged under the same conditions as experimental samples.