iGEM Measurement Kit

On this page you will find information on: The iGEM Measurement Kit

Introducing the Measurement Kit

Every lab has different equipment with different settings, and measurements of fluorescence or absorbance are often reported using arbitrary units (AU). These AU values from different labs cannot be directly compared. The iGEM Measurement kit contains resources that allow calibration of plate readers for fluorescent intensity and cell density measurements. Once these calibration protocols have been performed, you’ll be able to convert the arbitrary units you produce during your project into standard units. This will make your results much more powerful by making them directly comparable with those of other teams who have also calibrated their equipment.
Check out the 'What Is Measurement?' page to learn more about why comparable data is important for your research.

Converting OD₆₀₀ to Absolute Units

Cell density (i.e. how many cells are in a culture) is normally measured and reported using optical density (OD) readings. Whilst this approach can be used to provide a good estimate of cell density, the units reported are arbitary and can vary between equipment and labs. The measurement kit provides silica microspheres/beads which are similar in size and optical properties as bacterial cells. Therefore, 1 microspheres is approximately equal to one bacterial cell. By creating a serial dilution with known numbers of silica microspheres, a standard curve of number of microspheres against OD readings can be produced. Using this standard curve, OD readings for bacterial cell cultures can then be converted to an approximate number of cells, which is an absolute unit and comparable between equipment and labs.

The protocol for converting OD readings to absolute cell count measurements can be found here, along with spreadsheets to help with the required calculations.

Converting Fluorescent Intensity for Green/Yellow Fluorescent Proteins to Absolute Units

Fluorescent intensity (FI) is a measure for the amount of fluorescent protein present in a sample. FI is an arbitary reading, which means that it can not be accurately compared between different equipment and labs. The Measurement kit provides resources to convert FI to an approximate concentration of green/yellow fluorescent protein in a sample. Fluorescein is a fluorescent compound which has a similar fluorescent profile to GFPs and YFPs. A serial dilution with known amounts of fluorescein can be prepared to calibrate a piece of equipment's arbitary FI readings and convert them to concentration, which results in data which can be compared between labs and equipment. The method described above to measure number of cells should also be used so that concentration of fluorescent protein per cell can be reported.

The protocol for converting FI readings to absolute concentration measurements can be found here, along with spreadsheets to help with the required calculations.

Converting Fluorescent Intensity for Red Fluorescent Proteins to Absolute Units

Fluorescent intensity (FI) is a measure for the amount of fluorescent protein present in a sample. FI is an arbitary reading, which means that it can not be accurately compared between different equipment and labs. The Measurement kit provides resources to convert FI to an approximate concentration of red fluorescent protein in a sample. Texas Red is a fluorescent dye which has a similar fluorescent profile to RFPs. A serial dilution with known amounts of Texas Red can be prepared to calibrate a piece of equipment's arbitary FI readings and convert them to concentration, which results in data which can be compared between labs and equipment. The method described above to measure number of cells should also be used so that concentration of fluorescent protein per cell can be reported.

The protocol for converting FI readings to absolute concentration measurements can be found here, along with spreadsheets to help with the required calculations.