UNL 2018 Improving Early Detection of The Emerald Ash Borer

Interlab

What is the Interlab Study:

The iGEM Interlab study is a protocol that labs all across the world volunteer to complete and collect data from with the idea of improving synthetic biology with a specific goal. This year’s goal is to reduce variability in fluorescent measurements between labs in different regions by forming absolute units. The variability in bulk measurements and approximation of optical density (OD) reveals the need for a more consistent and reliable unit for fluorescent measurements.

LUDOX Calibration:

Calibration protocol were provided to ensure that we can take cell measurements under certain conditions and that we understand the process of the measurements. Standard OD600 measurements were taken without any pathlength correction feature to prevent any compromise to the accuracy of the measurement. LUDOX solution was provided and was used as a conversion factor. Our calibration results are listed below in Table 1.

	LUDOX CL-X	H20
Replicate 1	0.055	0.036
Replicate 2	0.055	0.036
Replicate 3	0.057	0.036
Replicate 4	0.060	0.036
Replicate 5	0.057	0.036
Replicate 6	0.021
Replicate 7	0.063
Replicate 8	3.036

Table 1: LUDOX Calibration Results

Particle Standard Curve- Microsphere Protocol:

The next step was to make a calibration curve for the particle concentrations when can be used to convert Abs600 to an estimated number of cells. Serial dilutions of a stock microsphere solution were completed in a well plate. Four separate serial dilutions were ran to help from a more accurate and reliable average concentration. Both linear and logarithmic graphs are included below in Figure 1 and 2 respectively.

Interlab Figure 1:

Interlab Figure 2:

Fluorescence Standard Curve- Fluorescein Protocol:

One cannot go plate reader to plate reader and compare results because of the arbitrary units used by all different instruments. The next step was to make a standard fluorescence curve so it is possible to compare results between teams. Serial dilutions of a stock fluorescein solution were prepared in a well plate and fluorescence was measured. Another four serial dilutions were completed to give a reliable average value.

Interlab Figure 3:

Interlab Figure 4:

Cell Measurement Protocol :

The cell preparation required for the measurement called for transformation of 8 different plasmids into E. Coli DH5 cells. Two colonies were picked for each plasmid and cultured. Overnight cultures were diluted to Abs600 of 0.02. Samples of each device were taken at the 0 hour and 6 hour marks. There were 8 devices total with 2 colonies per device and 2 measurement time points resulting in a total of 32 samples. Abs600 and fluorescence measurements were taken for all samples.

Team:UNebraska-Lincoln/InterLab