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.

Our Fluorescence Tracker is a hardware for potential users of our mutagenesis system to easily track whether the mutated protein has been expressed or not. Here we provide a step-by-step guide to using this hardware.

Step 1. Download and install the correct version of Micro-Manager from its official website according to your depending on your own computer setup (MacOS/Win-32/Win-64 are available). Older releases are recommended here.

Step 2. Double click on the Micro-Manager icon to start the software.

Step 3. Load Micro-Manager startup configuration file (.cfg files) that controls the camera and the Arduino board (the file openCV.cfg shown here only controls the camera). The file arduino_LED.cfg controls both the camera and the Arduino board.

The setup of our hardware is shown in the following picture. We developed the configuration script arduino_LED.cfg can control both the camera and the Arduino board. However, due to the lack of a filter glass, when projecting the light to the plate that has colonied by E.coli expressing fluorescence protein, the images recorded by the camera was far below expectation. Because of our inability to select an appropriate filter glass, we just propose a theoretically feasible hardware setup.

Step 5. After Step 4, a setting window will pop up. Tick the Time Points box to set how many pictures you want to take and the intervals of taking these pictures. Suppose we decide to record for 12 hours, we only need to type 144 in the text box behind Number and set 5 min in the text box behind Interval.

Step 6. Tick the Save images box to save the pictures to a local folder. Finally, click the Acquire! icon to start the automatic image acuiring process. Now you can see your plate if it is placed right under the camera.

**Step 8. If you want to access the recording device when you are not in the, you can use the software TeamViewer to connect your mobile phone and your computer, as is shown in the following figure. The picture is the screen shot from the mobile phone.

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.