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Abstract

Performing synthetic biology research requires the use of expensive equipment, which is problematic for underfunded laboratories in developing nations, new research facilities, and high school programs. There is a demand for low-cost, easily accessible laboratory equipment for manual centrifuging and plate reading. In order to address this need, an imaging device and an analyzing software app were designed to quantify the HSV color space from genetically recombined E.coli cells containing inducible chromoprotein constructs. The engineered chamber, Chrome-Q, captures controlled data by eliminating external light, which allows the Android compatible mobile app to calculate the HSV color space from predetermined RGB values. To inexpensively and efficiently pellet transformed cells, an existing 3-D-fuge design was optimized for integration with protocols developed for the Chrome-Q system. Proof of concept was achieved by the creation of an induction curve using three different chromoproteins from ATUM’s Protein Paintbox and the assembled genetic circuits: Promoter-R0040, Ribosomal Binding Site B0034, and Scrooge Orange/Virginia Violet/Tinsel Purple (TsPurple)-BBa_K1033906, using the Chrome-Q system. This project provides economically feasible devices and methods for underfunded research labs working with chromoprotein color expression.

Description

Purpose

When researching synthetic biology, underfunded labs often lack the monetary means to purchase expensive laboratory equipment; basic machinery, such as centrifuges, or more complex materials for analyzing data, such as plate readers, are commodities for many laboratories. The 2017 Lambert iGEM team decided to focus upon these issues, and, therefore, strives to make expensive equipment, like plate readers and centrifuges, more viable resources for underfunded labs.

Method

The approach Lambert iGEM used was multifaceted:
• Design a chamber to optimize and standardize conditions for detecting color
• Write a software application that can be used by anyone to quantify the color values using HSV
• Test and improve on a low-cost centrifuge
• Assemble and test these devices with chromoproteins under varying levels of IPTG induction

Using engineering design principles, the 2017 Lambert iGEM team aimed to develop an imaging device called the Chrome-Q to cost-effectively standardize conditions for quantifying chromoprotein data; additionally, the team improved upon the 3-D-fuge originally designed by the Prakash Lab to pellet cells at a low cost. Different chromoproteins - Tinsel Purple, Virginia Violet, and Scrooge Orange - were induced at varying levels of IPTG, causing the cells to express different intensities; pictures of the cells were taken using a Chrome-Q and were measured with a self-constructed software app that analyzes the pigments using HSV color space. Additionally, the team observed that a common issue encountered by synthetic biologists is the overlap of protein expression in the development of genetic circuits which negatively affects the quality of performance in a given cell. The 2017 Lambert iGEM Team worked to develop an inducible “switch” to further characterize ClpXP, a non-lysosomal proteolysis mechanism, with three Keio strains; the data showed a correlation between IPTG concentration and amount of protein degradation. Overall, Lambert iGEM desired to promote scientific research under any financial circumstance and to characterize non-lysosomal inducible protein degradation (CLiP’d).





Designed construct used to develop an inducible "switch"

Animation of ClpXp Protease

References

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Baker, T. A., & Sauer, R. T. (2011, June 27). ClpXP, an ATP-powered unfolding and protein-degradation machine. Retrieved Summer, 2017, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209554/

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Bhamla, M. Saad, et al. “Hand-Powered Ultralow-Cost Paper Centrifuge.” Nature News, Nature Publishing Group, 10 Jan. 2017, www.nature.com/articles/s41551-016-0009.

Burton , R.E. , Siddiqui , S.M. , Kim , Y.I. , Baker , T.A. , and Sauer , R.T. ( 2001 ) Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine . EMBO J 20 : 3092 –3100 .

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Lee C, Schwartz MP, Prakash S, Iwakura M, Matouschek A. ATP-Dependent Proteases Degrade Their Substrates by Processively Unraveling Them from the Degradation Signal.

McNerney, M. P., Watstein, D. M., & Styczynski, M. P. (2015). Precision metabolic engineering: The design of responsive, selective, and controllable metabolic systems. Metabolic Engineering, 31, 123–131. doi:10.1016/j.ymben.2015.06.011

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