Overview
To verify the function of our remote monitoring system for E. coli growth phases, we used step-by-step experiments to verify that our project is able to inform the E. coli growth phases to the user through the LineBot system. The proof of concept is divided into 3 parts, including phase indicator, monitoring device, and LineBot system. From the phase indicator, we combined a phase-specific promoter with a reporter gene to express distinct colors in desired phases. The function tests for the construct were performed by the fluorescent intensity and OD measurement. In the end, the ligation is performed to allow our indicator to differentiate either the mid-exponential phase, late exponential phase, or stationary phase.
Moving on to the monitoring systems, we combined the camera platform with deep learning and Google sheet to provide the automatic detection and recording system.
Finally, LineBot provides users with a notification system by retrieving data from Google Sheets. Users can enter a specific term and get the data easily and remotely.
We believe that “E.color'' is an integrated project preventing the time cost and personnel costs when performing the OD measurement. It is more than just a concept but feasible in the future.
Step 1: Phase Indicator
For the search of the phase-specific promoter, we come up with eight potential candidates according to mRNA expression profile. The fluorescence intensity as well as the OD value measurement are performed to determine the characteristic of those promoters. Among those, three promoters have shown phase-specific properties, the nirBDC promoter, which is the mid-exponential phase promoter; the glpABC promoter, which is the late exponential phase promoter, and the stationary phase promoter–hchA promoter. The constructs and the fluorescence expression are shown below.
Fig.1) The construct for nirBDC-mCerulean
Fig. 2) The construct for glpABC-mCherry
Fig. 3) The construct for hchA-AmilCP
Fig. 4) The fluorescent intensity and OD600 - time graph for nirBDC-mCerulean and glpABC-mCherry
Fig. 5) OD600 value - time graph for hchA-AmilCP
Fig. 6) The photo at 9hr for Fig. 5
Fig. 7) The photo at 10hr for Fig. 5
To enable E. coli to differentiate phases, three part ligation was performed. The confirmation was done by PCR with primers located at nirBDC promoter, glpABC promoter, and hchA promoter. The results are shown below.
Fig. 8) Electrophoresis result of PCR amplification
Then, the fluorescence intensity and OD measurement are performed again to determine whether the ligation would result in devastating effect. For amilCP measurement, the pictures are taken before and after the stationary phase. The results are shown in the graph below.
Fig. 9) The result for fluorescent intensity and OD measurement of ligation
Fig. 10) The photo of 10 hr to 11 hr from Fig. 9 to show AmilCP expression
Fig. 11) The photo of 10 hr to 11 hr from Fig. 9 to show AmilCP expression
Step 2: Monitoring Device
For verifying, we divided the monitoring system into two parts. First of all, to make sure that our detected phase is the correct phase, we compare the RGB color converter on the Internet with our DEVICE data on GOOGLE SHEET. This is to make sure that the RGB color detected on our model is the correct color since our phase detection on the colony is based on the RGB value. The result shows that our detected phase is the correct phase. Secondly, we start all the process by simply setting six plates on our 3D printing turnplates and activating the whole code to meet our title “Auto detection”. The process is recorded as part of the demo video in Device Page.
Fig. 12) The monitoring device
Fig. 13) Labeling for colony
Step 3: LineBot System
For the proof of the LineBot system, we discussed both the function of our LineBot system and the correctness of the data. First of all, to prove the LineBot retrieving function, we recorded the demo video. In the video, users can enter the keyword “Hint '' to search for the other keyword. The keywords include “website”, “phase”, “color”, and “google sheet”. For the brief information about the colony, users can either enter phase or color. The “color” will show the current RGB value of the colony, and the “phase” will show the percentage of colonies located in a specific phase. For more information, the “google sheet” could show the detailed data. Besides, by entering “website”, the user can directly link with our websites to know us better. For the demo video, please visit our Device page. Secondly, we compare the data on the Google Sheet with the data obtained from LineBot to confirm that the overall operation is consistent. This is to show the correctness of the data.
Fig. 14) Interface of LineBot
Fig. 15) Data retrieve by LineBot