Team:William and Mary/project

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Project

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OUR GOAL: The goal of this project was to create a set of tools that would open up the door to studying calcium dynamics to future teams. Calcium is a crucial signal in many aspects for eukaryotic organisms, including brain development and function; however, creating molecular tools to study calcium dynamics in eukaryotes is extremely difficult. By creating parts to emulate, measure, and record calcium spikes in model prokaryotes, our team hoped to pave the way for future teams to build genetic circuits for eukaryotic systems. We planned to create three different classes of parts that can: 1. Create calcium influx in bacteria
2. Indicate the presence of calcium
3. Keep track of calcium spikes and produce a reporter molecule after a certain number of spikes

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3. Tracking and reporting Ca2+ spikes:

A calcium-dependent promoter would activate a genetic counting circuit that would, after a specified number of spikes, produce a reporter gene to indicate that this number of spikes occurred in the cells.

1. Creating Ca2+ influx in bacteria:
The green algae Chlamydomonas naturally produces two non-specific cation channels that activate in response to particular wavelengths of light. Named channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2), we thought that these could be used to induce calcium spikes in prokaryotes. There exist multiple varieties of ChR1 and ChR2, which have been optimized for different ions and conditions. 2. Indicating presence of Ca2+:
Genetically Encoded Calcium Indicators (GECIs) are fusion proteins of a fluorescent protein (XFP) and calmodulin (calcium-binding messenger). XFP is initially in a non-fluorescing state. Upon binding of Ca2+, a conformational change in calmodulin activates the XFP, producing fluorescent light.

Future Directions: With the tools that have been developed and are continuing to be developed, the William & Mary iGEM team is making huge amounts of progress towards achieving our goals of inducing calcium spikes, reporting the presence of calcium, and counting calcium spikes in prokaryotes. We have adapted two channelrhodopsins to make them usable by iGEM teams, and have made great strides towards having a functioning RCaMP (or GCaMP) available to be used in future iGEM projects. We hope that opening up this door will create more options for future teams intending to study this important signaling molecule.

The Calcium Kids (and our marvelous PI!)

Click on the photo to look at our Parts page!

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