Team:Brasil-USP/Measurement
Measurement
Measuring Promoter Strength using a Camera + Gimp
Measuring Promoter Strength using a Camera + Gimp
Over ten thousand parts are cataloged in the Registry of Standard Parts! They are promoters, protein coding sequences, ribosome binding sites, plasmid backbones and many others part types. However, several parts have never been characterized or are poorly characterized. We propose a method to determine promoters strength just using a camera and a free software by measuring fluorescence GFP levels.
How it works?
All these results were obtained using high-end equipaments capable of precise and well calibrated measurements. Conversely, we wanted to make some measurements ourselves. We then wondered: what if we have only a commercial camera and no money to buy expensive software? Here is what we have done:
1- Took pictures with smooth and homogeneous luminosity, using a Canon EOS 600D. White backgrounds may help, but they do not seem to be necessary. Here is an example of such picture.
Figure 1 - Picture of one of our 96-wells plate. Devices 1, 2 and 3 are in wells A09 through B05.
2- Then we used GIMP, a free and open software considered by many a professional software for image processing, to show us how the distribution of scales of green. See below how it is done.
Figure 2 - Analyzing the green distribution with GIMP. To do so, first select (for selection tool, press r) the area you want to analyze. Then, open Window / Dockable Dialogues / Histogram. Finally, on the top corner of GIMP window, select the green channel.
3- Using the histogram dialogue, you have access the average values and standard deviations. All you have to do is compare.
By eye, it is almost impossible to distinguish among the different samples. Conversely, using the histogram dialogue you will be able to have a finer measure. For instance, our Device 1 (see Interlab Study) showed an average of \(160.2\), while the negative control was \(152.2\). Device 3 was \(152.6\), i.e., really close to our negative control (compare with Figure 3). Although it would be better to use the formula shown above, it is also a good definition of promoter strength the simple ratio of "fluorescences" - in our case, the average level of green. See in the table below our results for each of the devices:
Figure 3 - Results for promoter strength using a commercial camera and a free software (GIMP). Column "Average" shows the promoter strength (ratio of levels of green between a device and Device 1).
The method is not perfect, as you can see we have got the positive control twice as strong as we got with our equipament. However, we could see a clear distinction between devices 1, 2 and 3. Also notice that device 3 is really close to the negative control, as we observed in our experiments.
We are currently developing a 3D model of a support to hold a camera still while pictures are taken. This way, we could evaluate how the levels of green evolve through time. We believe a few adjustments are necessary to really make this method a competitive, low-resolution option for quick & dirty experiments. With this 3d model, everyone in the world will be able to simply print this model (nowadays, ABS is pretty cheap!) and be ready to make their own experiments.