Team:The Tech Museum/Project

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Overview:

We created a pool of plasmids designed to produce wide hue diversity in bacteria. Variation in promoter strength randomizes the relative expression levels of red, yellow, and cyan color reporters in each plasmid. In this way, we can create bacterial ‘pixels.’ Theoretically, the hue of each resulting colony should represent a particular combination of reporter protein concentrations, similar to how an RGB LED operates.

Museum visitors are guided through the transformation of e.coli with this plasmid pool to generate plates with a rainbow of bacteria colonies. Next, they take those petri dishes to an interactive scanning station. We developed software that uses digital imaging and computer vision to analyze the color, intensity, and rarity of the bacteria colonies on the visitor’s plate. A dynamic visualization of our team’s aggregate color data is then updated in real time with each participant's individual contribution to our iGEM team.

Do some promoter-color combinations always fail? Do others dominate? Are there colony hues that are never seen? Or, seen repeatedly? Our software and the participation of museum visitors is designed to find that out.

Details and Documentation:

Biology

To generate random color diversity in bacteria, we designed and had assembled libraries of tri-color plasmids (thanks to the generous sponsorship of DNA2.0). The base arrangement of plasmid functional elements was modeled on the three-color expression plasmid pZS2-123 designed by Cox et al. (2010). Our plasmids contain three different reporter proteins that are each controlled by combinatorially-inserted promoter-rbs pairs of varying strengths. Nine previously described promoter-rbs pairs that span a range of expression levels were selected from the literature (Kosuri et al. 2013; see notebook for specific sequences). When paired with the 3 reporters, the output of this assembly strategy is a plasmid pool with 729 unique combinations. Theoretically, each unique plasmid should drive a specific ratio of the three different colored reporter proteins to produce a defined hue.


In total, 4 plasmid pools were generated. Two reporter protein versions of the library were made: one with fluorescent proteins (PaprikaRFP, KringleYFP, CindyLouCFP) and one with chromogenic proteins (red, yellow, blue). Additionally, each of these was also made with both a low and high copy ORI. We tested and optimize all four of these plasmid pools in our visitor-accessible museum wetlab setup. After optimization of e. coli transformation and plating conditions, we determined that the low copy fluorescent plasmid pool was ideal, so it was used for all subsequent exhibit prototyping and data collection with museum visitors.


Software

As part of our combined iGEM project and museum exhibit, we developed two apps, RainbowReader and eColor, for analysis and quantification of bacteria colony colors. The source code for both is available on github.

Rainbow Reader is a meteor application that photographs and analyzes petri dishes containing visible bacterial colonies using OpenCFU, gphoto2, and an optional barcode scanner for sample tracking. It is powered by Meteor and Node.js, supplying a user interface in web browser. It connects by USB to a Motorola DS457 barcode scanner and gphoto2-compatible camera.








Rainbow Reader optionally sends data to eColor (seen in image below), a sister meteor app that presents live visualizations of the aggregated measurements.


Demo: RainbowReader Test Server; eColor Test Server


Requirements:

Usage:

install requirements, buy camera & usb scanner
clone repo
update server/lib/settings.js to disable opencfu, barcode scanner, and gphoto calls as neededset $METEOR_ENV as desired
start meteor (it will need to be restarted after initializing npm meteor package)
read the instruction manual;


Bacteria Photobooth:

Supplies:
UV light box
Camera
Tripod
Light blocking cloth



Results:

Using this hands-on exhibit, our iGEM team of museum visitors analyzed a total of 2674 colonies of bacteria on 61 different petri dishes. Together, we found a total of 324 unique colors! The aggregate data for bacteria color frequency and distribution looked like this:



References:

Cox, R. S., Dunlop, M. J., & Elowitz, M. B. (2010). A synthetic three-color scaffold for monitoring genetic regulation and noise. Journal of Biological Engineering, 4(1), 10. doi:10.1186/1754-1611-4-10

Geissmann, Q. (2013). OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PloS One, 8(2), e54072. doi:10.1371/journal.pone.0054072

Kosuri, S., Goodman, D. B., Cambray, G., Mutalik, V. K., Gao, Y., Arkin, A. P., … Church, G. M. (2013). Composability of regulatory sequences controlling transcription and translation in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 110(34), 14024–9. doi:10.1073/pnas.1301301110

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