Team:SCUT-FSE-CHINA/InterLab
InterLab
Abstract
As we all know, reliable and repeatable measurement is a key component to all engineering disciplines, so as synthetic biology. However, it is hard to do replication in different labs. We often used fluorescence as an indicator of promoter activity. However, it is difficult to compare fluorescence measured from different laboratories because the data are reported in difference units or shown in a different way.
The goal of this year’s iGEM interLab study established a GFP measurement protocal based on engineering principles that anyone with a plate reader can use in their lab, so teams attended can use the same exact protocal around the world to produce common, comparable units for measuring GFP with different plate readers, which are available in most labs to measure protein.
This year's iGEM Interlab study is to quantify expression of five different plasmids and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm for GFP expression per cell for all plasmids (or devices).
Protocol
We followed the protocol of iGEM InterLab measurement study to measure absorbance at 600 nm of LUDOX and made a FITC fluorescence standard curve.
As the very first step of the cell measurement experiment, we began by transforming the 8 plasmids (Positive Control, Negative Control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, and Test Device 6 - locations listed below) from Kit Plate 6 or Kit Plate 7 into E. coli DH5-alpha cells. Plasmids were purified from cultures of the positive colonies, for we could tell the fluorescence through the colonies. Next, we followed the plate reader protocol posted on iGEM InterLab measurement study, that can be found here . We tried many different setting for measuring fluorescence: different excitation and emission wavelengths, reading from the top or bottom of plate, and using filter or not. In the result section, we only show excitation 485nm and emission 538nm, reading from the top of plate without using the filter.
Material
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
Ps: we only used kit plate 6.
Instrument
Biotek SYNERGY neo2
Results
Figure 1. Fluorescein Standard Curve. This is the result of serial two-fold dilutions of FITC from the stock solution of 25μM. The value of R- squared is 0.9904.
Figure 2. Absorbance measured at 600nm over 6 hours for all devices.
Figure 3. Fluorescence over 6 hours for all devices.
Figure 4. The ratio of uM Fluorescein / OD600 values.
Discussion
Device 1 has shown the maximum fluorescence, followed by Device 2 and Device 4. Device 6 shows the lowest fluorescence, followed by Negative Control. Besides, we consider Device 1 has the strongest promoter, Device 6 has the weakest promoter, for their uM Fluorescein / OD600 are above two and almost zero respectively.
