The Green Fluorescent Protein (GFP) was used as a measuring tool to achieve repetitive measurements in laboratories around the world provide the standardized methodology for synthetic biology and offer a discipline that intersects biology and engineering. The purpose of this protocol is to provide researchers in each laboratory with effective programs and data analysis form to solve problems that cannot be directly compared to measured data values. The problem that the fifth interlab hopes to solve is the high variability between the OD (optical density) measured between laboratories, because in the method of normalization of fluorescence values, we need to divide the total fluorescence by the number of cells to determine the average expression level of GFP per cell. This experiment is dedicated to find a more straightforward way to determine the number of cells, such as by normalizing to absolute cell counts or colony forming units to reduce inter-laboratory variability in fluorescence measurements.
1. OD600 Reference point – LUDOX Use LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform your absorbance (Abs600 ) data from plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer.
2. Particle Standard Curve – Microsphere Prepare a serial dilution of monodisperse silica microspheres and measure the Abs600 in plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement will allow you to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.
3. Fluorescence standard curve – Fluorescein prepare a serial dilution of fluorescein in four replicates and measure the fluorescence in a 96 well plate in your plate reader. By measuring these in your plate reader, you will generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration.
transform Escherichia coli DH5α with DNA in selected wells in the Distribution Kit
Pick 2 colonies from each of the transformation plates and inoculate in 5mL LB medium withChloramphenicol. Growcells overnight (for 16 hours) at 37°C and 220 rpm
After a uniform dilution, each sample of 100μl per well was pipetted in a 96-well plate, and OD and fluorescence were measured at 0h and 6h post-cultured period.
Colony Forming Units per 0.1 of OD600 E. coli Count colonies for two Positive Control (BBa_I20270) cultures and two Negative Control (BBa_R0040) cultures.
Colony cytomrtry analysis