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PROJECT DESCRIPTION:

A high throughput device to advance optogenetics

Why advance optogenetics?

Optogenetics is a new research method allowing tight spatiotemporal control of intracellular protein activity using differing wavelengths of light. In the presence of light, proteins can dimerize, translocate, open membrane channels and go through a plethora of other responses. This demonstrates a novel way of studying biological processes and directing cell function for research and medical purposes. For example, by tying the function of light responsive systems like Cryptochrome 2 or pDawn to a protein of interest, one can activate cell signaling processes or gene transcription by simply shining blue light(Ohlendorf et al., 2012).

Our project was inspired by current advances in applying optogenetics to metabolic engineering, which is manipulating the cell’s metabolism pathways to alter product synthesis outcomes. Such manipulations can be carried out with light by controlling transcription of key metabolic enzymes with different patterns or doses of light to optimize production of certain metabolic products. For example, pDawn is an opto-tool that activates a gene of interest under light stimulation. The activity of the opto-tool can be assessed by engineering the opto-tool to promote the transcription of fluorescent proteins such as mAmetrine and GFP (Drobizhev et al., 2011). pDawn can be easily activated to different levels and at different times throughout a host cell's life cycle with variable light conditions. Exploring these parameters allows researchers to discover the optimal induction pattern for a desired product, thus maximizing yield.

What are current research methods?

The field of metabolic engineering has made great strides in pharmaceutical and biofuel production, and this research has recently been enhanced by optogenetic tools by allowing the timing and dose of metabolic enzyme expression to be controlled in a rapid and precise manner. Although the potential for optogenetics in metabolic engineering is high, progress is currently limited by a lack of methods to effectively sample the vast parameter space afforded by optogenetic control. Optogenetic experiments are currently low throughput and require frequent disruption to take measurements. Moreover, experiments are often limited to one experimental condition at a time because of limitations in illumination technology for cell culture. Although recently developed high-throughput optogenetic stimulation technology could be applied and coupled with commercial plate readers, real-time monitoring of high-thorughput optogenetic experiments requires constant user intervention, requires significant investment in commercial hardware, and disrupts stimulation protocols, making certain experiments, such as highly resolved time courses, impossible to perform

Our Solution: the OptoReader

The OptoReader was designed, engineered, and tested to overcome the current research barriers of optogenetics and increase the depth and types of experiments that can be carried out. Our team engineered an economical device that allows for high-throughput optogenetic experiments through simultaneous fluorescence and optical density readings and optogenetic stimulation in a 96-well plate format. The OptoReader consists of two printed circuit boards (PCB) that sandwich a 96-well plate. The top board is made up of an array of 192 LEDs used for opto-tool stimulation (96 LEDs) and optical density measurements (96 LEDs). The bottom PCB is made of a similar array of 96 LEDs that excite fluorophores and 96 photodiodes (light sensors) for measuring OD and fluorophore emission. With a user-friendly graphical user interface, the OptoReader’s 96-well format allows for a high-throughput experimental design where the user can apply up to 96 different stimulation patterns and simultaneously measure relevant cell outputs (OD and fluorescense) in real time.

See our project promotional video below for more!

Learn more about the engineering process here

ENGINEERING

References

1. Drobizhev, M., et al., Two-photon absorption properties of fluorescent proteins. Nat Methods, 2011. 8(5): p. 393-9.

2. Ohlendorf, R., et al. "From dusk till dawn: one-plasmid systems for light-regulated gene expression." Journal of molecular biology, 2012. 416(4): 534-542.

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