Team:NEU CHINA/Contribution
We recombined PmrCAB system to detect the spike protein of coronaviruses. The original PmrB ’s Fe (III) sensitive domains were replaced by the core binding domain of virus’ receptor, so that this two-component system can be sensitive to different virus’ spike protein.
See the Design page for the specific mechanism.
The three types of plasmids were constructed as follows(Fig. 1), and the corresponding part name isBBa_K3611002 , BBa_K3896007, BBa_K3896008.
Fig. 1 A: The plasmid was constructed to detect SARS-CoV-2’s spike protein(BBa_K3611002). B: The plasmid was constructed to detect MERS-CoV’s spike protein(BBa_K3896007). C: The plasmid was constructed to detect HCoV-229E’s spike protein(BBa_K3896008).
We have proved the effectiveness of the gene circuit(Fig. 1) through experiments. The engineered bacteria were induced by IPTG and then added spike protein to stimulate the engineered bacteria to express the reporter gene EGFP. As shown in the Fig. 2, the engineered bacteria induced by IPTG and stimulated by spike protein emitted distinct fluorescent signals.
Fig. 2 Fluorescence intensity of EGFP. Values represent the mean ± SEM. **p <0.01 versus control and ***p <0.001 versus control. Ctrl: Detection bacteria. IPTG: Detection bacteria + IPTG. IPTG+S Pr: Detection bacteria + IPTG + S protein.
This detection system can be more widely used in other virus detection besides SARS-CoV-2, MERS-CoV and HCoV-229E.
In order to improve the sensitivity of detection, we have improved the above parts by adding the quorum sensing system.
The system can enable the engineered bacteria stimulated by virus to transmit the detected signal to the engineered bacteria without virus protein through the signal molecule AHL secreted by LuxI, and finally the population expresses the reporter gene EGFP, making the detection result significant and avoiding false negative phenomenon.
See the Design page for the specific mechanism.
The gene circuit was designed as follows (Fig.3), and the improved part is BBa_K3896016.
Fig. 3 The two plasmids were constructed with pETDuet-1 and pACYCDuet-1 as the vectors, and were co-transformed to E.coli BL21(DE3) to verify the QS system.
To verify whether the quorum sensing system is effective, the following experiments were conducted.
Fig. 4 The fluorescence intensity of engineered bacteria without quorum sensing system and with quorum sensing.
With the addition of quorum sensing system, the detection sensitivity is improved, and the experimental results are more remarkable. It confirms that quorum sensing system can solve the false negative phenomenon caused by low virus concentration in the environment to a certain extent.
Learning that AHL can enter other cells after reaching a certain threshold, then combining with endogenous LuxR to form the co-complex, activating the corresponding promoter and expressing the reporter gene. We explored this threshold through experiments.
We used the following gene circuit(Fig. 5) and simulated the AHL secreted by bacteria through adding a series of concentration gradients AHL. The fluorescence intensity was then measured. As shown in Fig. 6, the fluorescence intensity in engineered bacteria with QS was enhanced at a AHL-dose dependent manner and step into plateau period when AHL reached 9 μmol/L.
Fig. 5 The plasmid was constructed with pACYCDuet-1 as the vector to explore the concentration threshold of AHL.
Fig. 6 The fluorescence intensity of engineered bacteria under different concentrations of AHL.
The results can be used as a reference for other iGEM teams.
Our hardware is based on air sampler and equipped with titration and detection system. The hardware physical diagram(Fig. 7) and application process are as follows.
Fig. 7 Physical view of the hardware.
Firstly, we use constant temperature constant current sampling. After enrichment, the samples are absorbed into different absorption bottles, which contain engineering bacterial solution that can detect different viruses induced by IPTG for 2h. Next, titrate the solution to be tested in the absorption bottle into a 24 well plate. Then, the liquid in the 24 well plate is detected by fluorescence, and it is hoped that the fluorescence signal can be analyzed by software, and the final detection result can be output. When the air sample contains the target virus, the engineering bacteria are stimulated to express the reporter gene EGFP and emit green fluorescence through a series of biological processes. If the fluorescence signal reaches the threshold, the alarm will be started. At the same time, we also use the micro camera for timing shooting to visualize the results. Finally, in order to ensure its safety and effectiveness, the hardware is equipped with a killing system to disinfect and replace the engineering bacterial solution at a certain time interval. See the Hardware page for specific design.
Therefore, our hardware has the functions of sampling, detection and sterilization, so as to realize safe multivirus detection.
Our hardware design has been recognized by relevant experts. They agreed that the design can be applied to the corresponding environment, such as airports, shopping malls and other places with large human traffic. We will carry out experiments to verify this later.