Team:LambertGA/Measurement


Measurement


Function

The ChromQ Light Chamber is a 3D-printed imaging measurement system used to quantify results of nutrient deficiency. For our current project, it is also used to quickly and inexpensively measure relative protein degradation through quantification of the color in chromoprotein expression.


Relative Color Quantification

Relative strength of the degradation of tsPurple cells (control, tsPurple DAS, and tsPurple LAA) can be measured by using a smartphone camera lens or any simple photography device (along with the color-controls for consistency).

Here are the steps for color quantification:
1. Place specimen* in box next to a control palette. Using a viewing opening at the top, adjust chamber to center specimen.
2. Using a smartphone camera, take a picture of the specimen, including the control palette. Be sure to focus the image so that all shades of the control palette can be visualized accurately.
3. Upload picture onto the website www.colorcodepicker.com.
4. Select color pixels of the target colonies.
5. The website will give an RGB value. Darker colors correspond to weaker relative degradation strength, while lighter colors correspond to stronger relative degradation strength. The RGB values of the results make up a spectrum that determines level of protein expression compared to intensity of color or RGB value.



*Specimen Preparation Protocol
1. If liquid culture, spin down pellet in centrifuge at 2450 x g for 10 minutes, decant excess liquid, and pipet approximately 5µL of pellet onto a square of parafilm.
2. If grown on agar plates, obtain a sterilized or disposable inoculation loop, take a couple of colonies or enough bacteria to form a clump approximately 3-5mm in diameter, and place onto a square of parafilm.



GFP Degradation Proof of Concept


The green gradient was generated from the RGB Color Gradient Maker created by PerBang and serves to show the range of GFP intensity in relation to its degradation level


Variation of color intensity and fluorescence in GFP degradation over the course of 60 minutes expressed in the vacuoles of protoplasts from Arabidopsis cells


Quantification of GFP degradation in relation to the relative intensity of vacuolar fluorescence - this is the model we aim to sophisticate in the future


Previous research has analyzed GFP fluorescence levels as it has been degraded. This degradation can be matched to RGB levels in the chart above to create a measure for quantifying GFP degradation with the ChromQ Light Chamber. This process can be similarly be applied to using RGB values to analyze degradation levels of chromoproteins.


Laboratory Controls

The two controls used for imaging will be the natural color of cells and solid black. The natural color of bacterial (E.coli) cells is not simply white, but is more of a creamy-white color. Degradation at the cream-white color is 0% degradation, while solid black is 100% degradation. A third control, ideally a shade of purple with 50% degradation, will be established.

Additionally, a color-control palette will be used for consistent imaging. As we realize that smartphone cameras tend to adjust color intensity, saturation, and brightness on its own in response to the colors present, the color-controls must always be present during imaging to balance the camera’s automatic adjustments.




As seen in the right image above, the color-control palette is composed of filters from Roscolux, which not only provide the RGB values as a color standard but also the wavelengths of the colors for further data analysis if desired. The yellow- and tan-toned filters on the top-left are designed to match the colors of cells that express no chromoproteins, or those that have completely undergone protein degradation. The darkest filters on the bottom-right will serve as a standard for cells expressing chromoproteins but have little or no degradation of protein. With additional experimentation, specific degradation percentages may be matched to the color and create a degradation spectrum based on color, much like the purple gradient depicted in the table on the left.



References

Dhakar, L. (n.d.). Image Color Picker (Z. A., Ed.). Retrieved October 10, 2016, from http://www.colorcodepicker.com/

Purple color codes. (n.d.). Retrieved October 10, 2016, from http://www.rapidtables.com/web/color/purple-color.htm

RGB Color Gradient Maker. (n.d.). Retrieved October 10, 2016, from http://www.perbang.dk/rgbgradient/

Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi, S., . . . Hara-Nishimura, I. (2003, September). Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. The Plant Journal, 35(4), 545-555. doi:10.1046/j.1365-313X.2003.01822.x

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