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A common issue perceived in underfunded labs across the globe is access to technology for biological analysis. The means often utilized for these analyses consists of exorbitant prices and requires transportation of samples to off-site locations to due equipment’s location. This lack of ready access has proven to be a hindrance, even to a number of iGEM Teams. In order to effectively address this issue, we designed a prototype to serve as an alternative for a plate reader and analyze bacterial samples for a low cost.

Chrome-Q

The evolution of Chrome-Q's from right to left

The 3-D modeling files can be found HERE if you would like to print the Chrome-Q.

This prototype, coined the “Chrome-Q”, is miniature light chamber that standardized the emission of light on bacterial pellets placed into wells engraved into the base of the dome, with measurements being taken through a phone’s camera at the top of the dome. Additionally, an app has been created to analyze the degradation of color as a progression, and measure the change in intensity and color as a value for the amount of protein degradation that has occurred. The app was designed to find samples using luminance (which is derived from RGB values). Then, the samples were analyzed in the HSV color model, mainly using Hue. Through a data curve obtained from various concentrations of cells, we have translated the expression of a certain color’s intensity as a reference point for percent degradation. In order to ensure consistency in the quantity of cells present at the time of measurement, we incorporated a microwell design into the base where cellular pellets can be placed.

Chrome-Q System Protocol

1. Make 5mL liquid cultures in triplicate of cumulative concentrations of IPTG in LB (0mM, 10uM, 100uM, 500uM, 1mM)
2. Make one trial (5 tubes) of control group containing 5mL of LB
3. Fill microcentrifuge with [(5 x #of experimental tubes) + 20] uL of diH2O (extra 20uL of diH2O accounts for unintentional error when inoculating liquid cultures)
4. Inoculate the microcentrifuge with a visible amount of cells from cultured plate (cells contain IPTG inducible sequence with color-expressive reporter)
5. Briefly (one or two “touches”) vortex microcentrifuge to suspend and evenly distribute cells in diH2O
6. Micropipet 5uL of suspended diH2O-cell mixture into each experimental tube (this step ensures a regulated amount of cells/ experimental tube)
7. Place in incubator at 37C for 24 hours
8. After growth period, centrifuge at 2500 RCF for ten minutes to obtain a pellet
9. Micropipet 200 uL of pellet into a PCR tube and centrifuge for two minutes in an electrically-powered centrifuge or 3 minutes in the 3D printed fuge
10. Vortex lightly (one or two “touches”) and Pipette up and down to resuspend cells.
11. Take 20uL from the center of the resuspended cells and transfer to a new PCR tube. (to continue with the 20uL, skip to the paragraph below; keep the remaining 180uL of cells for later directions).
12. Take 20uL from the center of the resuspend cells and transfer to a new PCR tube
13. Dilute 20uL of suspended cells from each tube in 80uL of H20.
14. Perform a serial dilution to get down to a .000X concentration. (There should be 4 dilutions).
15. Plate 20uL of cells from the final dilutions in a lawn in order to get individual countable colonies across the plate.
16. Grow cells in the incubator overnight.
17. Form a grid on the plate and use a random number generator to select a grid section.
18. Count the number of cells in the grid section.
19. Average the number of cells counted per microliter for each dilution and average the three. This will result in the average number of cells per microliter: the optical density.
20. Dividing the HSL values by the optical density results in the quantification measurements.
21. (Proceeding with the remaining 180uL of cells) Centrifuge the remaining 180uL of cells and plate 160uL of the supernatant. Avoid the pellet.
22. Pipette up and down in the remaining supernatant to resuspend.
23. Take 17uL of the sample and plate in triplicates.
24. Place the plates in the incubator (37C) for 24 hours
We performed three serial dilutions of 100uL of Tinsel Purple. We then plated 20uL of this on a quadrant of a plate. We determined the concentration of the cells was too great, due to the formation of lawns as opposed to countable colonies, and that a fourth dilution at a .000X will generate the most desirable growth. We also determined that each dilution did need to be plated in a lawn format on individual plates.

3-D Fuge

Our team member Gaurav demonstrating the 3-D Fuge.

As a continuation of the theme of low-cost technology, we contacted the Prakash Lab at Stanford University to obtain the design for the PaperFuge, and utilize its technology to obtain pellets efficiently. Dr. Saad Bhamla supplied us with the design files for the PaperFuge and several 3-D printed models Our team slightly modified the design to accommodate our needs, and optimize the necessary conditions for measurement. With the transfer of liquid cultures to PCR Tubes and spinning them through the 3-D Fuge (the updated Paperfuge), a distinct separation of LB media and cell pellets exists, and following aspiration of the media, the pellet can be successfully extracted.

Chrome-Q and 3-D fuge Synergy

Chromoproteins with varying levels of IPTG induction (ascending order) in the Chrome-Q wells. The first row is Tinsel Purple, and the third row is Scrooge Orange.

The pellet is then transferred into the wells present in the Chrome-Q, and analyzed using the desired phone. This overall design incorporates the necessity of quick biological analysis as well as a cost-efficiency to cater to the monetary restrictions experienced around the world. Through this, the Chrome-Q serves as an inexpensive yet accurate substitute for a plate reader while simultaneously being accessible and simple to use.