Description

At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the LUDOX Protocol in order to obtain a conversion factor to transform absorbance (Abs600) from the plate reader into a comparable OD600 measurement as would be obtained with a spectrophotometer. Next, we completed the Microsphere Protocol as it allows a standard curve of particle concentration which is used to convert Abs600 measurements to an estimated number of cells. Finally, by completing the Fluorescein Protocol we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Table 1).

Table 1. Parts received and tested during iGEM’s fifth InterLab Study

Table 1.

RESULTS AND DISCUSSION

1. MEASUREMENT OF LUDOX CL-X OD600 REFERENCE POINT

Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Table 2) which will be used in further Interlab study measurements.

Table 2. LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419

Table 2.

2. GRAPHING A SILICA MICROSPHERE ABSORBANCE (Abs600) STANDARD CURVE

Monodisperse silica microspheres exhibit size and optical characteristics similar to cells, with the additional benefit that the number of particles in a solution is known. Therefore, this measurement allowed us to construct a standard curve which can be used to convert Abs600 measurements to an estimated number of cells.

Fig. 1