Team:NEU CHINA/Proof Of Concept

Proof Of Concept

The human living environment is full of various microorganisms, bacteria, fungi, and of course viruses. From the Black Death to smallpox, from Ebola to SARS, it can be said that the struggle between humans and viruses has never stopped. In recent years, the coronavirus has attracted global attention, and a fast and effective detection method is an important means of interrupting the spread of viruses. This year, we built a multi-virus detection system and then used the hardware as a vehicle to make the system practically usable in the environment.

Overall design and validation

Hardware

Overall design and validation

In this project, we modify the PmrA/PmrB two-component system from Salmonella by replacing the original Fe(III) sensitive domain on the sensor protein PmrB with the coronavirus receptor core domain(Fig. 1), so that upon stimulation by coronavirus spike protein, a histidine kinase (HK) on PmrB undergoes autophosphorylation and subsequently transfers the phosphate group to the intracellular regulator conserved aspartate residues in PmrA, allowing PmrA to activate the PmrC promoter, which in turn expresses the downstream gene EGFP.

Fig. 1 The recombinant PmrCAB detection system.

In this project, we modify the PmrA/PmrB two-component system from Salmonella by replacing the original Fe(III)-sensitive domain on the sensor protein PmrB with the coronavirus receptor core domain(Fig. 1), so that upon stimulation by coronavirus spike protein, a histidine kinase (HK) on PmrB undergoes autophosphorylation and subsequently transfers the phosphate group to the intracellular regulator conserved aspartate residues in PmrA, allowing PmrA to activate the PmrC promoter, which in turn expresses the downstream gene EGFP.

LuxI/LuxR quorum sensing system was designed to improve the sensitivity of detection. This system allows engineered bacteria stimulated by viral signals to transmit signals via the signaling molecule AHL to other engineered bacteria not stimulated by viral proteins, so that they all express the reporter gene EGFP. Improvements to the system have resulted in more significant test results and fewer false positives. Please see the Design page for the specific mechanism.

In wet experiments, we placed the recombined PmrCAB system in E. coli BL21 (DE3) and extracted the receptor binding domain (RBD) of the coronavirus spike protein to stimulate the engineered bacteria.

The gene circuit(Fig. 2) and experimental results(Fig. 3) are as follows:

Fig. 2 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.

Fig. 3 The fluorescence intensity of engineered bacteria without quorum sensing system and with quorum sensing.

Hardware

For the actual environmental testing, we place the engineered bacterial solution in hardware that contains three systems for sampling, titration and detection, and also contains a kill function to ensure that it does not pose a threat to the environment or human health.

The working principle of this hardware is roughly as follows. Please see the Hardware page for the specific mechanism.

First, the air sample is collected based on the principle of an air sampler, and the sample is enriched and absorbed into a different absorption bulb. The bulb contains engineered bacterial solution that can detect different viruses after about 2 hours of IPTG induction. Our engineered bacteria are resistant to both ampicillin and chloramphenicol, so we add these two antibiotics to the medium to ensure the survival of engineered bacteria while preventing contamination by other bacteria.

Fig. 4 Sampling part of the hardware

Next, the solution in the absorption bulb is titrated separately into the 24 well culture plate.

Fig. 5 Titration part of the hardware

Then, the liquid in the 24 well culture plate is subjected to fluorescence detection, and the software is expected to analyze the fluorescence signal and finally output the detection results. When the air sample contains the target virus, the engineered bacteria are stimulated to express the reporter gene EGFP and emit green fluorescence through a series of biological processes. If the fluorescence signal reaches a threshold, the alarm will be activated. Meanwhile, a microscopic camera is used for timed filming to visualize the results.

Fig. 6 Detection part of the hardware

Finally, to ensure its safety and effectiveness, the hardware is retrofitted with a killing system to sterilize and replace the engineered bacterial solution at certain time intervals. Thus, our hardware is equipped with the functions of sampling, detection and killing all in one for safe multi-virus detection.

Current experiment data has verified the rationality of the design and the effectiveness of the detection, and the application of the hardware has been approved by the professionals. Next, we will combine the experiment with the hardware to test its application effect.