Team:NEU CHINA/Poster


Peixuan Zheng, Zihan Wu, Longfei Peng, Huili Li, Jinze Li, Yongye Huang, Chen Ding.

Traditional detection of environmental SARS-CoV-2 employs quantitative PCR method, with a minimum turnaround time of 6-hour. Here, we developed a rapid protocol, in which the presence of SARS-CoV-2 S protein is readily detected by genetically engineered ACE2-PmrA-PmrB system. In our work, we recombined the PmrA-PmrB two-component system derived from Salmonella to the surface of E. coli, and replaced the Fe (III) recognition site of PmrB with the core region of human angiotensin-converting enzyme 2 (ACE2),which could recognize the surface spike glycoprotein (S protein) of SARS-CoV-2. Upon recognition, the downstream signal pathway and the reporter gene are activated. Furthermore, an amplifier (hrp regulatory machinery) and extracytoplasmic function (ECF) σ factor were used to improve the sensitivity of the biosensor. A TEV protease-based post-translational regulation system to reduce the leakage is also used. In summary, our system shortens the time frame and reduces the cost and lab labors for detection.
Inspiration and Introduction
Current Detection of Coronavirus Have Disadvantages
The detection of coronavirus mainly based on two methods, testing the RNA of virus or the specific antibody. Quantitative Real-time PCR is one of the most common methods used to detect RNA. But RNA has poor stability, which leads to the high rate of detection missing, and high requirements for personnel and experimental sites. While the antigen-antibody detection can only test the sample from blood, leading to the limitation of its usage.
Comparison of viral RNA testing and antibody testing.

Virus detected by engineered bacteria
We modified the E. coli to make it capable of recognizing the S protein of SARS-CoV-2. PmrA-PmrB two-component system can accept the stimulation of the Fe (III) outside the Salmonella [1]. By replacing the Fe (III) binding domain with the core domain of ACE2, which is the receptor of virus’ S protein, after sensing the virus, EGFP will be expressed to output the fluorescent signal activated by the signal transduction pathway regulated by the modified PmrA-PmrB system.

Accessories to improve detection effeciency
In order to amplify the expression of the reporter gene when our engineered E. coli have detected the induction signal, we designed a cascade amplifier to enhance the output signal [2]. On the other hand, to avoid erroneous judgment of the experimental results caused by excessive background expression levels, we also designed a protein degradation system based on the TEV protease from Potyvirus, which can selectively reduce the reporter gene expression when no induction [3]. The diagram of "SARS detector".

[1] Marc M.S.M Wösten et al. "A Signal Transduction System that Responds to Extracellular Iron". 103.1(2000):113-125.
[2] Xinyi Wan et al. "Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals". 15.5(2019):540-548.
[3] Jesus, Fernandez Rodriguez, and C. A. Voigt. "Post-translational control of genetic circuits using Potyvirus proteases." Nucleic Acids Research 13(2016):6493-6502.
Detection Part

Upon sensing SARS-CoV-2, the sensor kinase PmrB autophosphorylates a highly conserved histidine residue and subsequently transfers the phosphoryl group to a conserved aspartate residue in its cognate response regulator PmrA. Then the phosphorylated PmrA binds to the promoter PmrC sequence to active the expression of the reporter gene egfp. We designed a bacteria co-culture experiment. Three groups of bacteria were constructed: engineered bacteria with detection function, prey bacteria expressing S protein and empty vectors. After being cultivated at 37℃ for 1h to make their OD600 reach 0.4~0.6, IPTG was used to induce. Strong green fluorescence can be obviously noticed at the group of detection bacteria and prey. And other two groups have less or even no fluorescent signal.

Fluorescence microscopy photos of detection bacteria. EV, bacteria with empty vector. The S protein is the Thr333-Gly526 of spike protein of SARS-CoV-2, which were expressed by E. coli BL21.
Cascade Amplifier
We hope the detection limit of our detection system is low enough meanwhile the output level is high enough to ensure trace viruses would not be missed. We designed a cascade amplifier to increase the output signal and lower the detection limit.

The biological amplifier. This device can amplify the transcriptional signal and the output level.

The cascade amplifier consists with two independent orthogonal amplifiers arranged in series, which was constructed using hrp operon from Pseudomonas syringae, and extracytoplasmic function (ECF) σ factor ECF11_987 and promoter ECF11_3726 from Vibrio parahaemolyticus respectively.

The two amplifiers were connected in series. The first amplifier’s output is the second one’s input, so that the second amplifier could amplify the first one’s output signal. In general, the output signal will be amplified twice.

The cascade amplifier and its characterization graph. A, Further amplifying the output of the reporter fluorescent signal by using an extra transcriptional amplifier based on ECFs. The amplifier parts are represented by triangles in the figure, while the hrp amplifier in orange and ECF in blue. B, Cells are induced by 5 varying concentrates of IPTG (0, 10-6, 10-5, 10-4, 10-3 mmol/L) after 1h.

We used protease-based post-transcriptional degradation regulation to lower the basal background. A protein degradation tag (AAV) was added to the reporter protein to reduce its expression. To obtain low basal level without sacrificing the high output, we next added a TEV proteas-based reporter protein degradation control system. The TEVp will be activated to be expressed by the same transduction input signal as amplifier, which can cleave the linker between the expressed reporter EGFP and its fused AAV tag.

The architecture of the low-leakage cascade amplifier. A cascade transcriptional amplifier designed on the basis of the hrp regulatory network and ECFs. The basal output level will be lowered by the TEV protease-based protein degradation control system.
Cell Wall-Deficient Bacteria
Now, the virus needs to cross the cell wall so that can touch the recombinant protein. If we remove the cell wall, the virus would touch the receptor directly and make the detection faster.

We used L-type hypertonic medium and cefixime to induce L-form bacteria. The microscopic examination after Gram staining showed that the bacteria have features of L-form bacteria. And there are two pictures showing the colonies. The left one shows the normal colony, and the right one is the L-form bacteria colony.

A, Colonies of normal engineered bacteria growing on plate medium. B, Colonies of L-form engineered bacteria after induction of hypertonic medium.
Biosafety Carrier
In order to achieve the primary practicality of the project, we chose double crosslinked hydrogel as carrier of engineered bacteria. Polyvinyl alcohol (PVA) - sodium alginate (SA) double crosslinked hydrogel has been proved to have good biocompatibility and could be a good carrier for engineered bacteria. The crosslinked hydrogel has good pore structure and mechanical strength. In this way, handling the engineered bacteria into the hydrogel would make sure them would not easily spread, and maintain the good ability of detecting virus at the same time.

The schematic of SA/PVA double crosslinked hydrogel. After the crosslinking, the PVA gel and SA gel molecules would penetrate and combine with each other. The pores are big enough to let the bacteria and virus through, while the mechanical properties are still well performed.


The SA/PVA double crosslinked hydrogel and the biocompatibility fluorescence microscopy photos. A, Hydrogel embedding engineered bacteria in it was putted in a glass bottle. In this way the bacteria have a carrier that is not easy to leak. B, Fluorescence microscopy photos of E. coli in the SA/PVA double crosslinked hydrogel, which can express enhanced green fluorescent protein when there is IPTG. The test group was inducted by the IPTG in concentration of 1mmol/L for 2h, while the control group without any induction.
Diasbled Caring

Sign language vedio recording

With the principle of humanitarianism, we want to make contributions to the disabled. We popularized sign language knowledge relating COVID-19 for special groups so that they can communicate efficiently in the current situation. Most of the hearing-impaired groups can only communicate with people by sign language, and they also have to accept the information of pandemic. In the process of pandemic prevention and control, many new nouns have never appeared in sign language system. The COVID-19 sign language expression is the first. In addition, we also introduced other sign languages for the hearing-impaired about the COVID-19. The community thanks us for this initiative and sent us a letter for thanks.
Mental Health Caring
At the same time, we also focused on the emotional and psychological changes of people under pandemic stress. Whether suffering from COVID-19 or anxious ordinary people, they all may leave a psychological and spiritual shadow to some extent, causing pressure on the future life. We went into the community tried to comfort these people and held a COVID-19 mental health counselling conference.

COVID-19 Mental Health Counselling Conference
Educational Resources Balance
In order to balance the educational resources in China, we conducted distance course through internet for children in Yunnan province, who are eager for scientific knowledge.

Distance Course
Nursing Home Caring
In September, the pandemic situation has been stable. We went to the community and schools, even kindergartens and nursing home, to give public speeches, bring them knowledge and accompany them through their daily lives.

Nursing Home
Thanks for Prof. Chen Ding and Dr. Yongye Huang for giving us guidance of the project.
Thanks for Renhong Medicine Co., LTD, Huaxia Medicine Co., LTD, Weifang Minsheng Hospital.
Shenyang Dance Finger Technology Co., Ltd for supporting our human practice.