Team:Lubbock TTU/InterLab
InterLab Study 2018
Measurement techniques are essential for quantifying and communicating results that are meaningful in comparison to a standard. The iGEM Measurement Committee aims to develop robust measurement procedures for enhancing the quantification and reproducibility of results through the InterLab Study. One of the most common measurement tools in synthetic biology is fluorescence, and although fluorescence data can be a reliable way to characterize constructs, it can be difficult to compare fluorescence data from different labs. This is due to variations in the units used to report fluorescence data, as well as the methods used to process this data. By participating in the 2018 iGEM InterLab Study, we hope to contribute measurement data that can be used to standardize fluorescence readings between labs using a 96-well plate. The results can then be used to effectively characterize, troubleshoot, and share biological constructs. The goal of the 2018 iGEM InterLab Study is to answer the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? To answer this question, participating iGEM teams follow a standardized protocol that involves measuring green fluorescent protein (GFP) expression in E. coli DH5alpha. By utilizing a series of plate reader calibrations, we were able to construct standard curves to relate Abs600 to OD600, concentration of silica microspheres to cell concentration, concentration of fluorescein to cellular GFP concentration, and CFU count to OD600 (which will ideally allow us to determine the approximate total cell number). We are using green fluorescent protein (GFP) because it is one of the most used fluorescent markers in synthetic biology, and as a result, most labs are equipped to measure it. We submitted data regarding the absorbance and optical density of cells expressing 8 different plasmids. Below you will find the data we collected for the InterLab Study using a Synergy Neo2 plate reader.Protocol
The specific protocol used for this InterLab study can be found here.Calibration
Calibration 1: OD600 Reference point- LUDOX Protocol
The first part of the protocol uses LUDUX CL-X as a reference point to convert our Abs600 readings from the plate reader to a comparable OD600 measurement. This is necessary because the path length in a spectrophotometer is standard (as determined by the width of the cuvette), while the path length in plate reader measurements is dependent on volume.
We obtained the following data for this calibration:
Calibration 2: Particle Standard Curve- Microsphere Protocol
The second part of the protocol uses monodisperse silica microspheres, which have sizes and optical properties similar to cells, to construct a standard curve that can be used to approximate cell number based on absorbance measurements.
We obtained the following data for this calibration:
Calibration 3: Fluorescence Standard Curve- Fluorescein Protocol
The third part of the protocol uses a serial dilution of fluorescein to create a standard curve to convert fluorescence readings from cells expressing GFP to protein concentration. We use fluorescein because it is cheaper than GFP and has a similar excitation and emission properties.
We obtained the following data for this calibration:
Calibration 2: Particle Standard Curve- Microsphere Protocol
The second part of the protocol uses monodisperse silica microspheres, which have sizes and optical properties similar to cells, to construct a standard curve that can be used to approximate cell number based on absorbance measurements.
We obtained the following data for this calibration:
Calibration 3: Fluorescence Standard Curve- Fluorescein Protocol
The third part of the protocol uses a serial dilution of fluorescein to create a standard curve to convert fluorescence readings from cells expressing GFP to protein concentration. We use fluorescein because it is cheaper than GFP and has a similar excitation and emission properties.
We obtained the following data for this calibration:
Transformation and Results
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Transformation of Test Devices:
We transformed the 8 shipping devices into chemically competent DH5alpha, plated them, and incubated overnight at 37C.
Cell Measurement
After growing liquid cultures of our transformed cells, we measured the Abs600 and fluorescence of the cells expressing each of the 8 devices at 0 hours and at 6 hours. We performed the dilutions and set up our 96-well plate as explained in the protocol, and obtained the following results:
Abs600, 0 Hours
Fluorescence, 0 Hours
Abs600, 6 Hours
Fluorescence, 6 Hours
Colony Forming Units per 0.1 OD600 E. coli Cultures
In the final part of the protocol, we used our plate reader to estimate colony forming unit (CFU) counts from OD600 readings. We set up the well plate, performed a dilution series, and plated cells all according to the protocol. Our results are detailed below:
