To track bacteria growth on the plate and observe the fluorescence recovery from nonsense mutation due to continuous mutagenesis, we devised this hardware - the Fluorescence Tracker. It provides continuous, hands-off recording of the growth of plate colonies as well as fluorescent protein expression. For users of our mutagenesis system, with the help of our hardware, they could plate, and then monitor all plates together to increase the likelihood of spotting bacteria colonies with recovered fluorescence at the earliest time point. After discussions 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 fluorescence recovery, it could be easily modified to monitor bacteria colonies growing out of any antibiotic plate. Our hardware allows us to come to lab knowing that a plate with desired colonies is waiting for us.
Motivation
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 might not be high enough, we prefer 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 experiments 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 the computer and is controlled by 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 the mobile App from TeamViewer which view the computer on your mobile phone, you can have access to view the fluorescence of any plates anywhere and anytime.
Demonstration
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). Old 1.4 releases are recommended.
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. Both cfg files are available upon request.
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. Due to lacking a motorized drive, we are not able to switch between appropriate filter glasss, what we shown here could be improved.
Step 4. Click on the Multi-D Acq. icon to open multi-dimensional acquisition window.
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 7. Click on the green magnifier icon to zoom in for a better view.
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.
Future work
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.