Team:Austin LASA/InterLab
h(g.Page, {title: 'InterLab', prev: 'https://2018.igem.org/Team:Austin_LASA/Experiments', next: 'https://2018.igem.org/Team:Austin_LASA/Attributions', selector: [2, 4]}, h('p', null, 'The purpose of the 2018 iGEM Interlab, in which we participated and submitted our results to iGEM, is to reduce variability in fluorescence measurements by correlating fluorescence to colony forming units rather than OD. In order to achieve this goal we conducted the following experiments as specified by the iGEM 2018 InterLab Study Protocol.'), h(g.Image, {src: 'https://static.igem.org/mediawiki/2018/9/98/T--Austin_LASA--OD600.png', position: 'center'}, h('p', null, h('b', null, 'Figure 1.'), ' Calibration 1: OD600 Reference Point') ), h('p', null, 'A conversion factor between Abs600 and OD600 is required because Abs600 is defined by the depth of the water which may vary slightly from well to well, whereas the width of a cuvette, which determines OD600 is constant. In order to calculate this conversion factor we created a standard curve of varying concentrations on Ludox CL-X as a reference point (Figure 1).'), h(g.Image, {src: 'https://static.igem.org/mediawiki/2018/0/00/T--Austin_LASA--ParticleStd.png', position: 'center'}, h('p', null, h('b', null, 'Figure 2.'), ' Calibration 2: Particle Standard Curve') ), h('p', null, 'Silica microspheres are comparable in size and optical properties to cells, and there is a known amount of particles per volume. Therefore, creating a standard curve of Abs600 over a dilution series of silica microspheres provides an estimate of the number of cells in a solution based on Abs600 (Figure 2).'), h(g.Image, {src: 'https://static.igem.org/mediawiki/2018/1/16/T--Austin_LASA--FluorStd.png', position: 'center'}, h('p', null, h('b', null, 'Figure 3.'), ' Calibration 3: Fluorescence Standard Curve') ), h('p', null, 'Plate readers report fluorescence in arbitrary units which vary between machines. So so in order to compare fluorescence values between different labs, fluorescence needs to be correlated with concentration of fluorescent molecule. To do this we created a standard curve of fluorescence of fluorescein, a comparable stand-in for GFP, over 4 replicate series of dilutions (Figure 3).'), h(g.Image, {embed: true, position: 'center', src: 'https://static.igem.org/mediawiki/2018/e/e2/T--Austin_LASA--FperOD.pdf', height: '500px'}, h('p', null, h('b', null, 'Figure 4.'), ' Cell Measurement') ), h('p', null, 'In order to normalize the fluorescence to the concentration of cells in a solution, we created a standard curve by growing up cells expressing GFP to a fixed concentration in LB media and measuring fluorescence along a series of dilutions (Figure 4). The preparation and transformation of our competent bacteria do differ from those recommended by iGEM because we used TOP10 and transformed using the Thermo Fisher TOP10 transformation protocol because we have had prior success in transforming small quantities of DNA with it. Initially we attempted to perform the transformation using the Zymo Z-Comp Transformation Protocol but only a few transformations were successful due to the low quantities of DNA being transformed.'), h(g.Image, {src: 'https://static.igem.org/mediawiki/2018/e/e1/T--Austin_LASA--FperP.pdf', position: 'center', embed: true, height: '500px'}, h('p', null, h('b', null, 'Figure 5.'), ' Colony Forming Units per 0.1 OD600 E. Coli Cultures') ), h('p', null, 'In order to achieve the overall goal of normalizing fluorescence values to CFU we diluted a starting culture to 0.1 OD600 so that it would be within the linear range of the plate reader and then diluted that by factors of 8 x 10^4, 8 x 10^5, and 8 x 10^6, then plated those dilutions, and counted the number of colonies formed on the plates. Multiplying the number of colonies by the final dilution factor yielded CFU per milliliter (Figure 5).') );