Tracks/Measurement/Interlab study/Plate Reader Overview
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Introduction
The objective of this year’s iGEM Interlab study is to quantify expression of five different reporter constructs. These constructs have GFP under the control of different promoter and ribosome binding sequences (RBS). A key development over last year’s study is to evaluate the expression in absolute units.
Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). While this is an indirect measurement, it provides a useful insight into expression levels and has the significant advantage that it can be monitored continuously without disrupting cells.
Because we are really interested in understanding the expression behavior within individual cells, it is also necessary to have a measure of the total population. This is routinely done using OD600. This is not an ideal proxy for cell numbers, but it is easy to measure and again can be done in a non-disruptive manner. Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.
One key limitation of both fluorescence and OD600 is that they are both relative measurements. That is they are dependent on the instrument used and the settings of that instrument on that particular day. So fluorescence measured on two different instruments will give different value: even if they are of the exact same make and model differences will arise due to variation in lamp intensity and, precise alignment of the optical path, performance of the detector etc.
To transform fluorescence from a relative unit (e.g. counts per second) into an absolute unit (molecules of GFP), it is necessary to calibrate the instrument response. We aim to do this using the supplied FITC as a standard reference material. You will measure the fluorescence of your instrument using a dilution series of this reference material to construct a standard curve. We have previously performed this standard curve on our own instrument alongside a standard curve for purified GFP. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.
Regarding measurements of cell number, OD600 is a relative measurement because it is a measure of light scattering and is not a true absorption event. Therefore the light falling on the detector is dependent on the physical geometry of the machine (size of detector, physical distance etc). Furthermore there is a non-linearity to these measurements as cell density increases, as multiple scattering events can occur and photons can be scattered back into the detector path. Calibration events require complicated experiments based on flow cytometry that do not lend themselves to calibration by standard curves. It is therefore not possible to transform OD600 measurements to an absolute value of cell number. However, we aim to control for instrument variability, at least to some degree, by measuring a standard scattering solution of a mono-dispersed silica suspension (LUDOX). The objective is to see if a simple, single fixed-point measurement can be used as a ratiometric adjustment to provide greater uniformity in fluorescence/OD600 measurements across sites.
Method Overview
Measurements can be made in whatever spectrophotometer and fluorimeter you have in your lab. A plate reader with a 96 well format is ideal as it provides a convenient format for multiple measurements. However, single cuvette based machines can equally be used, and in many cases will give more sensitive readings, but you will get through a lot of cuvettes (if you use disposable ones), or do a lot of cuvette washing (if you use glass cuvettes). The methods are written from the perspective of using a 96 well format, but if you are using cuvettes just adjust and either do dilutions directly into disposable cuvettes, or into tubes prior to measurement. If re-using cuvettes it is good practice to start from low fluorescence/ absorbance samples up to high to reduce chances of carry-over.
The first objective is to obtain your standard curves for fluorescence based on the FITC reference material provided. The standard curve must be obtained under EXACTLY the same instrument conditions that you will use when you do your cell based expression assays. This includes all settings that affect the amplitude of the signal collected: filters or monochromator settings; slit widths; gain settings; plates or cuvette type used; measurement from top or bottom (in plates); number of reads (integration time); orbital averaging (available in some plate readers). You may not know right now what settings will be suitable when you do your cell based assays. The objective is to fix the obvious settings now, such as monochromator/ filter, plate and cuvette type, top or bottom reads. The key settings that affect sensitivity are slit width and/or gain. You MUST therefore collect now several standard curves under different sensitivity settings.
When doing cell based assays you should as far as possible settle on a single setting, but this may not be possible as it may not have sufficient dynamic range. By having a series of standard curves collected with different sensitivity you can use one (of a limited number) of the settings to obtain appropriate data (i.e. within range on the instrument). Because you will have a standard curve to match this setting, you can still transform into absolute units.
