Team:NEU CHINA/Design
Fig. 1 Schematic diagram of the overall design of the project.
Several coronavirus outbreaks have had many adverse effects on human beings. Conventional nucleic acid detection and antibody detection have high requirements for experiment equipment. We design a simple environmental multi-virus detection system, hoping to contribute to the fight against coronavirus.
In this project, we choose E. coli as the chassis to construct engineered bacteria for detecting coronavirus. Due to the similarity of infection mechanisms, the SARS-CoV-2, MERS-CoV and HCoV-229E are firstly selected as targets to assessed the feasibility of the multivirus detection system. The design includes unprecedented detection system modified with the PmrA/PmrB two-component system, LuxI/LuxR quorum sensing system with Hrp amplifier to enhance the sensitivity of the system, and a device to enable the application. Different types of engineered bacteria are constructed to make them sensitive to their specific target coronavirus. By putting three of them in the same device, multivirus detection would be realized. Moreover, with LuxI/LuxR quorum sensing system from E. coli used to improve the virus sample detection threshold, and the Hrp amplifier from Pseudomonas to amplify the output signal, the expression of reporter gene, the device we built can quickly, accurately and conveniently catch the virus in the sample.
After critical comparison, the PmrCAB system is selected for virus detection.
PmrA/PmrB is a two-component regulatory system in Salmonella, which originally is a regulatory system sensitive to Fe (III) [1]. The transmembrane sensor protein PmrB contains a histidine kinase (HK) domain in the intracellular segment, which will self-phosphorylate as soon as sensing extracellular signals, and subsequently transfers the phosphate group to the conserved aspartate residue in the intracellular response regulator PmrA. Phosphorylated PmrA becomes the activator of PmrC promoter to activate downstream genes. The original PmrB ’s Fe (III) sensitive domains were replaced by the core binding domain of different virus’ receptor, so that this two-component system can be sensitive to different virus’ spike protein.
Fig. 2 The recombinant PmrCAB detection system.
The target of detection is viral S protein, the monomer of homotrimeric spike glycoprotein on the envelope of almost all the coronavirus. It is an essential part of the virus in binding to sundry human cell receptor, fusing virus’ envelope with the cell membrane, mediating viral infection.
The S1 subunit of SARS-CoV-2’ s spike protein is responsible for recognizing human cell Angiotensin-converting enzyme 2 (ACE2) [2]. It is the part which virus used to sensing target cell then fuse its own envelope with the cell membrane to achieve infection. It was found that the core binding domain of ACE2 is concentrated between Thr20 and Asn90, while the RBD of S protein is Thr333 to Gly526 of its S1 subunit. Therefore, the polypeptide sequence of ACE2 fragment was used as the core detection binding domain.
S protein on MERS-CoV envelope can bind to human cell dipeptidyl peptidase 4 (hDPP4) [3]. MERS-CoV fused its own envelope with the cell membrane in a similar way to SARS-CoV-2 after recognizing. Therefore, we determine that the core binding domain of hDPP4 is located between Gly260 and Asp330, and the RBD of MERS’ S protein is Gln471 to Asp580. The MERS-CoV detection bacteria are constructed as the same way as mention before.
The receptor of HCoV-229E is human aminopeptidase N (hAPN). Core domain of hAPN is Ala281 to Gly330, and S protein RBD is Lys201 to Ser321 [4]. HCoV-229E’ s detection bacteria constructed also in the same method.
Fig. 3 Receptor binding domain of SARS-CoV-2 and core binding domain of ACE2
Fig. 4 Receptor binding domain of MERS-CoV and core binding domain of DPP4
Fig. 5 Receptor binding domain of HCoV-229E and core binding domain of hAPNAfter being stimulated by extracellular virus’ spike protein, a series of intracellular reactions would activate the expression of the reporter gene EGFP.
Fig. 6 A: The plasmid was constructed to detect SARS-CoV-2’s spike protein. B: The plasmid was constructed to detect MERS-CoV’s spike protein. C: The plasmid was constructed to detect HCoV-229E’s spike protein
Considering the low virus concentration in the actual environment, only a few of engineered bacteria can detect the virus and express EGFP with low output signal in general.
Therefore, quorum sensing system is used to let the first batch of activated bacteria transfer the activation signal from individual one to the population.
Fig. 7 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.
Fuqua first proposed the concept of "quorum sensing" (QS) in 1994. The concept is used to describe the way of specific communication in bacteria at population size [5]. Through this mechanism, bacteria play different biological functions in the form of multicellular, produce signal molecules and release them into the environment; When the signal molecules in the environment reach a certain threshold concentration, they induce the expression of specific genes dependent on cell density in bacteria, so that bacteria show new behavioral characteristics on the population scale, such as bioluminescence, extracellular polysaccharide formation and so on.
In the acyl homoserine lactones (AHL) mediated quorum sensing system composed of LuxI / LuxR, LuxI in bacteria is responsible for the catalytic synthesis of signal molecule AHL, and LuxR is activated as a receptor for this signaling molecule and forms the co-complex LuxR-AHL, which activates the corresponding promoter to regulate the expression of downstream genes [6].
In our design, when the engineered bacteria recognize the virus, PmrC initiates the expression of the downstream gene LuxI and LuxR. Catalyzed by LuxI, AHL starts to be synthesized continuously and part of it penetrates the cell membrane to the extracellular. When the concentration of extracellular AHL reaches a threshold, it re-enters the cell and combines with LuxR to form a LuxR-AHL co-complex, the co-complex can activate the corresponding promoter Plux_HS and express the reporter gene EGFP.
By adding the quorum sensing system. When a few engineered bacteria detect the virus, the information can be transmitted to other engineered bacteria through the mediation of signal molecule AHL, and finally the population expresses the reporter gene EGFP, making the detection result significant and avoiding false negative phenomenon.
We hope to improve the sensitivity of our detection system to ensure the fluorescence sensor would not miss the positive signal. Therefore, the Hrp amplifier, a transcriptional signal amplifier, is applied.
Fig. 8 The two plasmids were designed with pETDuet-1 and pACYCDuet-1 as the vectors to test the efficiency of the Hrp amplifier.
The natural hrp regulatory network contains activator HrpR and HrpS, which would form an ultrasensitive co-complex HrpRS, the activator of hrpL promoter, after expression. The network has been proven to amplify the output transcriptional signal very much.
[1] Zhang, S. , Han, D. , Ding, Z. , Wang, X. , Zhao, D. , & Hu, Y. , et al. (2019). Fabrication and characterization of one interpenetrating network hydrogel based on sodium alginate and polyvinyl alcohol. Journal of Wuhan University of Technology--Materials Science Edition.
[2] Sa, A. , Aa, A. , Vv, B. , & Mr, A. . (2020). Fabrication and characterization of a novel polyvinyl alcohol-graphene oxide-sodium alginate nanocomposite hydrogel blended pes nanofiltration membrane for improved water purification - sciencedirect. Separation and Purification Technology, 250.
[3] Wang, N. , Shi, X. , Jiang, L. , Zhang, S. , Wang, D. , & Tong, P. , et al. (2013). Structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4. Cell Research, 23(008), 986-993.
[4]Crossley, B. M., Mock, R. E., Callison, S. A., & Hietala, S. K. (2012). Identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229E. Viruses, 4(12), 3689–3700.
[5] Hauk, P. , Stephens, K. , Virgile, C. , Vanarsdale, E. , & Bentley, W. E. . (2020). Homologous quorum sensing regulatory circuit (hqsrc): a dual-input genetic controller for modulating quorum sensing (qs)-mediated protein expression in e. coli. ACS Synthetic Biology, XXXX(XXX).
[6] Galloway, W. , Hodgkinson, J. T. , Bowden, S. D. , Welch, M. , & Spring, D. R. . (2011). Quorum sensing in gram-negative bacteria: small-molecule modulation of ahl and ai-2 quorum sensing pathways. Chemical Reviews, 111(1), 28.