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Country codes are only necessary when calling telephone numbers in other countries. These are dialled before the telephone number with the international dialling prefix (or the + symbol) in the country from which the call is placed. For example, if you are calling from the US to Mexico, the US international dialling prefix is 011, so the US caller would dial 011, then the Mexico country code, and then the phone number.
Measurements of GFP fluorescence were calibrated using serial dilution of fluorescein with PBS in quadruplicate, using the protocol from ref. 9, as illustrated in Fig. 1b. Starting with a known concentration of fluorescein in PBS means that there is a known number of fluorescein molecules per well. The number of molecules per arbitrary fluorescence unit can then be estimated by dividing the expected number of molecules in each well by the measured fluorescence for the well; a similar computation can be made for concentration.
Calibration to colony-forming units (CFU), illustrated in Fig. 1c: Four overnight cultures (two each of positive and negative controls), were sampled in triplicate, each sample diluted to 0.1 OD, then serially diluted, and the final three dilutions spread onto bacterial culture plates for incubation and colony counting (a total of 36 plates per team). The number of CFU per OD per mL is estimated by multiplying colony count by dilution multiple. This protocol has the advantage of being well established and insensitive to non-viable cells and debris, but the disadvantages of an unclear number of cells per CFU, potentially high statistical variability when the number of colonies is low, and being labor intensive.
Comparison with serial dilution of silica microspheres, illustrated in Fig. 1e: This new protocol, inspired by the relationship between particle size, count, and OD7, uses quadruplicate serial dilution protocol of 0.961-μm-diameter monodisperse silica microspheres in water, similar to fluorescein dilution, but with different materials. These particles are selected to match the approximate volume and optical properties of E. coli, with the particles having a refractive index of 1.4 (per manufacturer specification) and typical E. coli ranging from 1.33 to 1.417. With a known starting concentration of particles, the number of particles per OD600 unit is estimated by dividing the expected number of particles in each well by the measured OD for the well. This protocol has the advantages of low cost and of directly mapping between particles and OD, but the disadvantage that the microspheres tend to settle and are freeze-sensitive.
Reliably determining the number of cells in a liquid culture has remained a challenge in biology for decades. For the field of synthetic biology, which seeks to engineer based on standardized biological measurements, it was critical to find a solution to this challenge. Here, we have compared the most common method for calibrating OD to cell number (calculation of CFU) to two alternative methods of calibration: LUDOX/water and microsphere serial dilution. The qualitative and quantitative benefits and drawbacks of these three methods for OD calibration are summarized in Table 1.
Each team providing flow cytometry data also obtained their own sample of SpheroTech RCP-30-5A Rainbow Calibration Particles (SpheroTech). A sample of this material is a mixture of particles with eight levels of fluorescence, which should appear as up to eight peaks (typically some are lost to saturation on the instrument). Teams used various different lots, reporting the lot number to allow selection of the appropriate manufacturer-supplied quantification for each peak. 2ff7e9595c
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