Team:Washington/ECOLIGHTTUNE

From 2013.igem.org

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Revision as of 04:06, 22 September 2013

Contents

Backgrounds

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The goal for this app is to make a standardized system for light control without the common problems many of the current ad hoc light projection systems come with: high cost, difficult assembly, and low reproducibility. With mobile device tablets integrating into our everyday lives, we found it fitting to develop a program to take advantage of their pervasiveness and utilize them as synthetic biology instruments.

E. colight was a tablet application designed by our iGEM 2012 team in order to project light onto bacterial cultures. This year, our team wanted to continue our 2012 project by expanding the functionality of our app, calibrate it using existing light-inducible promoter systems, and determine the maximum number of experiments we can perform on a single tablet. By designing experiments with high reproducibility that explores the strengths and limitations of the app, we hope to create the canonical system for optogenetics testing.

In addition to characterizing in depth the use of the app, we also developed new features that can accommodate a broader range of growth vessels, a vibration feature, and utilize the heat output of the processor as an incubator.

App

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E colight has been designed to shine different wavelengths of light on growing bacterial cultures. The app was designed and tested on the Samsung Galaxy Tab 7.7 which has an Super AMOLED display. Each pixel on an Super AMOLED display is made up of three LEDs corresponding to each of the colors in the RGB color format. This format is advantageous for the green light inducible system because the green output of the display is monochromatic in that only the green LEDs are turned on. Furthermore, the RGB LEDs output spectrum are very compatible with published red [1], green [2], and blue [3] light gene inducers. Another advantage of the display allows for a “true black”, which emits no light and minimizes background effects [old - in that no light is emitted which would allow for the lowest level of background] in a light inducible system. Finally, development of our app in the Android operating system allows for the easiest distribution of our app to actual users due to its open source nature, lower relative cost of Android mobile devices, and accessibility of the Google Play Store.

-Close up picture of Super AMOLED screen http://en.wikipedia.org/wiki/AMOLED

One our primary goals was to make this app flexible and useful for multiple microbial growth formats. The app was designed for experiments that use standard sized petri dishes, 60 mm petri dishes, and 96-well plates for both liquid and solid phase media. Each light-emitting unit can be controlled individually, which allows for multiple experiments to be performed simultaneously. In the case of the 96-well plate, this means each well can be controlled individually, which potentially allows for 96 simultaneous yet different experiments. In addition to these default settings, the diameters of the wells can also be adjusted to the user’s current experimental setup as well as the intensity and wavelength of the light. Samples can be exposed to mixtures of wavelengths through a blinking option which cycles through the RGB colors.

"FIGURE REQUEST:  Light spectral data"

We implemented a vibration setting with the intention of agitating liquid cultures to promote growth. Though this feature is fully functional in the current release, users are advised against extended periods of activation as the hardware cannot sustain high intensity vibration without failure. For this reason, the tablets were placed on orbital shakers to provide the agitation required for rapid growth.

"FIGURE REQUESTS:"

- screenshots

- video demo


Methods

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To characterize the app, we tested the output of GFP in response to light by using a tablet running E. colight and working light systems in 96-well plates or small petri dishes [size/photos?]. We used our green light-induced GFP and red light-repressed GFP systems. After inoculation into M9 media, the cultures were incubated under the appropriate light conditions on the tablet for eight hours. The amount of GFP was then quantified using a plate reader to measure the relative fluorescence.

As the tablet releases heat as it runs, it could potentially act as an incubator. We encountered this capability when our culturing temperatures were higher than expected. We realized that the processor in the tablet was generating heat in addition to the heat supplied by the incubator. However, this bug could potentially be a feature to sustain bacterial growth. With just a tablet, light tests could be done in any typical lab setting.

Early into testing, we realized that the heat from the surface of the tablet in a 37C incubator caused bacterial growth to slow and media to evaporate quickly. We realized that the processor in the tablet was generating heat in addition to the heat supplied by the incubator. The temperature increase was compensated for by elevating the plates using an empty petri dish and by lowering the ambient temperature in the incubator to 30C. Using this new setup, cultures grew normally.

While the heat released from the tablet presented a problem when using an incubator, it could potentially be exploited as a feature to sustain bacterial growth. To test this theory, E. coli containing the green light-induced GFP system on agar plates were grown on the tablet under green or dark light conditions. The temperature of the top of the tablet was measured as XYZ, and plates had to be incubated for XYZ hours as opposed to 8 hours in the incubator to see the same amount of GFP expression. This shows that, with just a tablet, light tests could be done in any typical lab setting.

We tested the light bleedthrough between adjacent wells in a 96 well plate by illuminating three wells sequentially and comparing the GFP output of cultures growing in the lit wells to the adjacent growing wells (Figure X). Other wells completely surrounded by dark (D5-E11) or light wells (D2, E2) were used as controls. The fluorescence of a “dark” well next to a “light” well was up to 60% of the original fluorescence (Figure Y); as a result, we recommend leaving one well between different light conditions.


Results Summary

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