Team:UC San Diego/InterLab
Introduction
This year, UCSD iGEM team actively participated in the Fourth International InterLab Measurement Study. The purpose of this year’s InterLab study is to encourage teams all over the world to follow the same protocol and produce common and comparable measurements for Green Fluorescence Protein, to standardize the protocols . In this way, the data obtained from the multiple teams can help iGEM researchers to compare the variability of fluorescence activity with high level of accuracy.
Method
Transformation
The first step of the interlab study was to transform the following 8 plasmid (Positive Control, Negative Control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, and Test Device 6 ) into E. coli DH5-alpha cells. The kit plate 6 was used.
- Positive Control (BBa_I20270): well 20B
- Negative Control (BBa_R0040): well 20D
- Test Device 1 (BBa_J364000): well 20F
- Test Device 2 (BBa_J364001): well 20H
- Test Device 3 (BBa_J364002): well 20J
- Test Device 4 (BBa_J364003): well 20L
- Test Device 5 (BBa_J364004): well 20N
- Test Device 6 (BBa_J364005): well 20P
Table 1. Kit plate 6 construction
Measurement of LUDOX-S40 as the Reference point
LUDOX-S40 was used as a single point reference to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement.
Table 2. Measurement of LUDOX-S40 and Water (Gold cells are calculated values)
Serial Dilution for Fluorescein Fluorescence Standard Curve
Accurate pipette 100 µl of PBS and 200 µl of fluorescein 1x stock to obtain a 96-well-plate serial dilution, from which the standard curve of fluorescence for fluorescein concentration was obtained. 1-12 are the data obtained from serial dilution.
Figure 1. Schematics for the Serial Dilution
Table 3. Serial Dilution of Fluorescein
Figure 2. Fluorescence vs. Fluorescein Concentration
Figure 3. Fluorescence vs. Fluorescein Concentration in a log scale
The standard curve (log scale) follows a roughly linear trend.
Measurement of OD600
2 colonies from each transformed plate were selected and inoculated on 10 mL LB Medium+Chloramphenicol. The cells grew 18 hours at 37°C and 220 rpm. The overnight culture was measured under OD600.
500 µL samples from each of the 8 devices of the cultures at 0, 2, 4, and 6 hours of incubation were taken and put on ice. There are two colonies per device, for a total of 16 samples per time point.
Figure 4. Plate Pattern
Table 4. Raw Plate Reader Measurement of Two Colonies from Four Time Points
Table 5. Raw Fluorescence Measurement of Two Colonies from Four Time Points
