Difference between revisions of "Team:IIT Delhi/InterLab"

The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work. In the previous studies, we showed that by measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule, we can greatly reduce the variability in measurements between labs. However, when we take bulk measurements of a population of cells (such as with a plate reader), there is still a large source of variability in these measurements: the number of cells in the sample. Because the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, we need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Usually we do this by measuring the absorbance of light at 600nm, from which we compute the “optical density (OD)” of the sample as an approximation of the number of cells. OD measurements are subject to high variability between labs, however, and it is unclear how good of an approximation an OD measurement actually is. If we used a more direct method to determine the cell count in each sample, then potentially we could remove another source of variability in our measurements. Converting between absorbance of cells to absorbance of a known concentration of beads. Absorbance measurements use the way that a sample of cells in liquid scatter light in order to approximate the concentration of cells in the sample. In this year’s Measurement Kit, we provide you with a sample containing silica beads that are roughly the same size and shape as a typical E. coli cell, so that it should scatter light in a similar way. Because we know the concentration of the beads, we can convert each lab’s absorbance measurements into a universal, standard “equivalent concentration of beads” measurement. 2. Counting colony-forming units (CFUs) from the sample. A simple way to determine the number of cells in a sample of liquid media is to pour some out on a plate and see how many colonies grow on the plate. Since each colony begins as a single cell (for cells that do not stick together), we can determine how many live cells were in the volume of media that we plated out and obtain a cell concentration for our sample as a whole. We will have you determine the number of CFUs in positive and negative control samples in order to compute a conversion factor from absorbance to CFU.