Design
We want to create a device that can detect the concentration of a given pathogeny, which requires a relationship between the concentration of the virus in the wastewater and the intensity of the fluorescence signal we producing. First, we have to build some “hands” that hold pathogenic microbes
Construction of random library of yeast two-hybrid system
Yeast two-hybrid system is a hybrid system for detecting protein-protein interactions. Yeast two-hybrid analysis system is based on the principle that many eukaryotic transcriptional activators consist of two physically separate and functionally independent domains, namely DNA-binding domain (DNA-BD) and transcriptional activation (AD) . The former can recognize and bind to specific nucleotide sequences (UAS) in the promoter of the target gene, while the latter activates the transcription of downstream target genes. If the two domains are separated by molecular biological techniques and expressed in the same host cell, the transcription of the response gene can not be activated.

Firstly,I designed and synthesized a random DNA fragment encoding 20 peptides
Secondly, the fragment was constructed on the primary vector pDONR207 by GATEWAYBP reaction, then the primary library plasmid (pDONR207-random petide) was obtained, and the successful pDONR207-random peptide was subsequently constructed for LR reaction with the pGADT7-GW vector, finally, the secondary library plasmid (pGADT7 GW-random peptide) was obtained, and the secondary library plasmid was then transferred into the yeast Y187 strain, at which point the random library construction was completed.

The design of hACE2 SOE PCR
Overlap Extension PCR(SOE PCR) is a technique for obtaining single point mutant genes, which is a site-directed mutagenesis technique in vitro. Using specific primers with complementary ends, the two PCR products were made into overlapping chains, and in the next round of amplification, the different fragments were spliced by overlapping chain extension. The technique does not require Restriction enzyme or ligases, nor is it restricted by the location and type of mutations. Mutations can be inserted at any point in the DNA. Overlap Extension PCR has been widely used in life science. To find the target protein, we used computational biology based on ACE2 evolution to predict sites with stronger binding to the spike protein. We used the UCSF CHIMERA to simulate the structure of hACE2 based on the RBD with SARS-CoV-2. The key AA interacting with RBM in HACE2 are K31, E35, D38, M82, and K353. Among them, K31 and K353 in HACE2 are the most critical residues for RBM recognition. According to the existing literature, the amino acids that interact with human in SARS-COV-2 are L455, F486, Q493, S494, N501 and Y505. By mutating the amino acids at the key sites, we can determine whether the space between the key atoms is smaller (smaller means they are more strongly bound) , and we can further verify this inference by calculating the binding energy between the two atoms.

Escape model
CheZ
CheZ is a protein derived from Enterobacterium that can influence the behavior of E. coli by phosphorylating CheY. CheY is a key regulator of flagellar motility response, and when it is phosphorylated, it causes the flagella to spin clockwise, and the phosphate protein phosphatase CheZ dephosphorylates CheY, causing the flagella to spin counterclockwise [4]. Studies have shown that when CheZ is knocked out of the chemotaxis pathway, the tumbling of E. coli cells predominates, while the overexpression of CheZ inhibits the tumbling of [5] and causes the high-speed linear movement of E. coli.
EL222
At the same time, with the help of Jilin University, we decided to use EL222, a light control element, which is a bacterium from the bacterium Li-Ray Erythrobacteriace HTCC2594's blue light sensory protein. It consists of a light-oxygen-voltage (LOV) domain that acts as half of the light-sensing domain at the N-terminus, and a LuxR-type spiral-turn-helix (HTH) DNA-binding domain, which acts as an effector domain in the C-terminal half. In the dark, EL222's interaction with DNA is negligible, and when light is activated, DNA binding is significantly enhanced. The DNA sequence of EL222-binding motifs is shown as 5'-RGNYWWRGNCY-3′ (Y = C or T, W = A or T, R = A or G, N = any nucleotide). When the domain is in the dark, the LOV domain non-covalently binds flavin single nucleotides (FMN) to chromophores. Under light, it forms a covalent adduct with nearby cysteine residues, which trigger conformational changes in proteins and transmit light signals. EL222 exists in the dark as a monomer. When blue light is irradiated, EL222 binds to each other to form a dimer that interacts with DNA and stimulates the expression of downstream genes. [6]
We couple this light-control gene with CheZ and a toxic protein so that under the exposure of blue light, E. coli will accelerate its movement due to overexpression of CheZ here. At the same time, the suicide gene that promotes autolysis of the bacteria will also begin to be expressed, but its toxicity is not enough to make the bacteria lyse immediately, and our "courier" will look for the area without blue light at high speed during the "countdown to death", once it enters the area without blue light. The bacteria will immediately "brake" and the suicide gene will stop expression. Our "courier" can successfully reach the area without blue light with its "cargo". The result is an increase in the concentration of virus in the blue-free region and almost virus-free in the blue-light area

Detection System
PMRA/PMRB is a two-component regulatory system for salmonella, which is originally sensitive to Fe (III)[7] . The transmembrane sensor protein PMRB contains a histidine kinase (HK-RRB- domain in the intracellular segment, which is self-phosphorylated upon sensing an extracellular marker, the phosphate group is then transferred to the conserved L-Aspartic Acid residues of the intracellular response regulator PMRA. Phosphorylated PMRA becomes an activator of the PMRC promoter to activate downstream genes. The original FE (III)-sensitive domain of the PMRB was replaced by the core-binding domain of different viral receptors, allowing this two-component system to be sensitive to the spike proteins of different viruses We modified the engineered bacteria provided by Tsinghua, which had been transferred to a two-component system, to sense the external material in the region of the“Tentacle” sequence that we had previously made using a random peptide library. In the presence of external markers, the two-component system will stimulate a series of reactions resulting in a certain fluorescence signal.

Reference
[1] Yang E,Zha J, Jochel J,etal- Bad, a heterodimeric partner for Bcl-xL and Bcl-2,displaces bax and promotes cell death [J]· Cell, 1995,80( 27) :285- 291.
[2] Yang M ,Wu Z, Fields S· Protein- peptide interact ions analyzed with the yeast two-hybrid system [J]· Nucleic Acids Research, 1995,23( 7) :1152- 1156.
[3]Clontech In· Matchmaker gal4 two hybrid sysytem3, 1999:PT3247-1. http://www·clontech·com/tech info/ manuals/PDF/ PT 3247-1. pdf
[4]M.D. Baker, P.M. Wolanin, J.B. Stock, Signal transduction in bacterial chemotaxis (in English), Bioessays 28 (1) (Jan2006) 9–22.
[5]S.C. Kuo, D.E. Koshland Jr., Roles of cheY and cheZ gene products in controlling flagellar rotation in bacterial 3147Blue Light-Directed Cell Migration chemotaxis of Escherichia coli (in English), J. Bacteriol. 169(3) (Mar 1987) 1307–1314.
[6]Takakado A, Nakasone Y, Terazima M. Sequential DNA Binding and Dimerization Processes of the Photosensory Protein EL222. Biochemistry. 2018 Mar 13;57(10):1603-1610. doi: 10.1021/acs.biochem.7b01206. Epub 2018 Feb 20. PMID: 29432690.
[7]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.
[8]Horton RM, Cai ZL, Ho SN, Pease LR. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 1990 May;8(5):528-35. PMID: 2357375.
[9]Luan J, Lu Y, Jin X, Zhang L. Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection. Biochem Biophys Res Commun. 2020 May 21;526(1):165-169. doi: 10.1016/j.bbrc.2020.03.047. Epub 2020 Mar 19. PMID: 32201080; PMCID: PMC7102515







