To track bacteria growth on the plate and observe the recovery of fluorescence due to continuous mutagenesis of the starting mutated protein, we devised this hardware - the Fluorescence Tracker. It provides continuous, hands-off recording of the growth of bacteria on the plate as well as fluorescent protein expression. For users of our mutagenesis system, with the help of our hardware, they could plate, and then monitor quite a number of plates to increase the likelihood of spotting bacteria colonies with recovered fluorescence at the earliest time point. Through the discussion with our PI, we improved our hardware by adding remote access through TeamViewer, which allows visualizing the dynamic changes on smartphones. Although the current hardware is only suitable for monitoring the recovery of fluorescence, it could be easily modified to monitor bacteria colonies growing out of any antibiotic plate. Our hardware allows us every time going to the lab will have a plate with desired colonies waiting for us.
In our experimental setup, we decided to demonstrate whether desired mutations of our gene of interest occurs by viewing whether the stop codon ahead of the real stop codon of the mRNA of eGFP is mutated into a non-stop codon. This means that after removing IPTG and aTc from the bacteria that have been used to induce mutation, we should coat the plate and keep track on the color of each colony that forms on the plate. However, as the mutation rate of our system is not high enough, we need to coat quite a few plates to increase the probability that at least 1 colony can express eGFP. Finally, we should put them under the fluorescence microscope one by one. Those steps render the demonstration step laborious. As a result, we built this Fluorescence Tracker hardware to automatically track the fluorescence change with cell growth. The Fluorescence Tracker is composed of a camera and an Arduino board connected to your computer and is based on a software called Micro-Manager. The Arduino board is connected to a flashlight that can emit R/G/B light to stimulate the fluorescence of eGFP. The camera is used to record the cell growth and the change in fluorescence. Finally, using a mobile App called TeamViewer which can link your mobile phone with your computer, you can have access to viewing the fluorescence of any plates anywhere and anytime.
User guideline and demonstration
At present, we just proved that our recording setup could work properly. However, for lack of critical devices, we haven't come up with a good solution to record the fluorescence. Moreover, this system can only work for one plate, but there will be a lot of plates that need to be tracked in R-Evolution. As a result, we should further build a device that can record many plates with high resolution.