iGEM 2022 — Team fafu

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Collaborate throughout the year with at least one other 2022 iGEM team on a set of shared objectives related to both of your projects.{% endblock %}
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# NOTEBOOK

[April](#p-1)
[May](#p-2)
[June](#p-3)
[July](#p-4)
[August](#p-5)
[September](#p-6)
[October](#p-7)

| NOTEBOOK | | | | |
| --- | --- | --- | --- | --- |
|  | Team | Experiments | Human Practice | Model |
| --- | --- | --- | --- | --- |
| April | | | | |
| --- | --- | --- | --- | --- |
| 1 | Diversified team formation | Laboratory safety and security seminar | Exploration of the project context |  |
| 2 |  | Experiment design | Designing Human Practice Closed Loop |  |
| 3 |  | Selecting topics | Establishing a public platform of WeChat |  |
| 4 |  |  | OUC-China's first group meeting about the aspect of art |  |
| 5 |  |  | Met with BIT, planned the partnership route and started the first online meeting |  |
| May | | | | |
| --- | --- | --- | --- | --- |
| 1 | Member Interview | Project Product Design | Exploration of the project context | Discussing hardware design ideas with BIT |
| 2 |  | Exploring experiment methods | FAFU's first offline meeting to discuss direction and potential of the project | Hardware required for functionality and design |
| 3 |  | Lab members identified and lab safety training | First project seminar with BIT to share HP experience to BIT |  |
| June | | | | |
| --- | --- | --- | --- | --- |
| 1 | Three days offline seminar which all FAFU members join in at Dragon Boat Festival | Refined the experimental design plan | Human social practice questionnaire writing and preliminarily distributing | Second project seminar with BIT, BIT shares Hardware experience to FAFU |
| 2 | Promotion video script Writing | Initial access to the laboratory | Conceptualize innovative Education programs and how to implement them effectively |  |
| 3 | Determine the first version of the project logo and event poster | Get acquainted with Tsinghua and share information about two-component system | Improve the information of the public propaganda platform, open a column for disinformation, and intermittently update the biological news and the progress of the team |  |
| 4 |  |  | Planning rural education activities |  |
| July | | | | |
| --- | --- | --- | --- | --- |
| 1 | Modify the team logo, and the team flag |  | Meeting with OUC-China group to discuss the direction of art cooperation | Determined hardware solution and start 3D modeling |
| 2 | Presenting projects at China iGEM Online Meetup | Well-designed random peptide library | Travel to Xiamen University to communicate with XMU-China team |  |
| 3 |  |  | Travel to Pingtan for rural education |  |
| 4 |  |  | Update WeChat public platform |  |
| 5 |  |  | Advancing the work of Human Practice |  |
| 6 |  |  | Invited BIT, JLU-China, XMU-China, OUC-China, JIANGNAN-CHINA to draw synthetic biology cartoons |  |
| 7 |  |  | Tsinghua invited us to co-organize an online public lecture on synthetic biology, and invited eight iGEM teams from China to deliver the lecture |  |
| August | | | | |
| --- | --- | --- | --- | --- |
| 1 | Maked a poster, presented the project and the poster at 9th CCiC | SOE PCE experiments were conducted simultaneously | Update WeChat public platform | Completed the first version of hardware design and first visit to Jinshan wastewater treatment plant for research |
| 2 | Complete promotion video shooting and collect ideas for Presentation Video shooting |  | Advancing the work of Human Practice |  |
| 3 |  |  | The third group meeting with BIT to monitor each other's project progress and improve the work of Human Practice |  |
| 4 |  |  | Communication with NEU-China |  |
| September | | | | |
| --- | --- | --- | --- | --- |
| 1 | Preliminary wiki design | Setting out in randomized peptide library | Planning campus orienteering run | Hardware 3D printing successfully and field |
| 2 | experiment proceeds | Yeast two-hybrid experiment conducted |  | trips |
| 3 |  | Organizing the results of the project | Planning the 2023 iGEM Recruitment Lecture |  |
| 4 |  | Update WeChat public platform |  |  |
| October | | | | |
| --- | --- | --- | --- | --- |
| 1 | Update wiki |  | 2023 FAFU-iGEMer Recruitment Lecture |  |
| 2 |  |  | Update Synthetic Biology online comic at WeChat |  |
| 3 |  |  | Orienteering |  |

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

# Engineering Success

[SOE](#p-1)
[Random Peptide Library](#p-2)
[PmrCAB Two-component system](#p-3)
[Suicide part](#p-4)
[the Escape Part](#p-5)

## SOE

### Design

We used a model to predict eight point mutant sequences with stronger interactions with the RBD structural domain of the new coronavirus by bioinformatics on the original sequence of ACE2.

#### Table 1.ACE2 point mutation predicted enhancement sites

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Number | Spike binding sites | ACE2 | Distance change before and after mutation | Binding energy | Predictions |
| 1 | SARS-CoV-2 | hACE2 | Original sequence | -14.38 | / |
| 2 | Q493 | l\_21\_V | 6.541->6.506 | -14.15 | Stronger |
| 3 | F486 | K\_26\_R | 5.132->3.8007 | -14.38 | Stronger |
| 4 | F486 | T\_27\_A | 9.886->9.387 | -14.39 | Stronger |
| 5 | L455 | K\_31\_R | 13.985->11.889 | -14.37 | stronger |
| 6 | F486 | N\_33\_I | 8.529->8.309 | -14.37 | Stronger |
| 7 | F486 | H\_34\_R | 15.184->12.349 | -14.36 | Stronger |
| 8 | F486 | E\_37\_K | 15.175->10.415 | -14.35 | Stronger |
| 9 | F486 | T\_92\_I | 7.047->7.0247 | -14.38 | Stronger |

### Construct

Then we had the company build the original Ace2 sequence we designed on the pGADT7 vector, and synthesize the primers we designed for point mutation, and then mutate the original ACE2 sequence.

![](https://static.igem.wiki/teams/4253/wiki/enginneer-success/2.jpg)

Figure1. pGADT7-ace2

### Testing

After getting the plasmid constructed by the company, we mutated the synthesized pGADT7-ace2 which was combined with primers, and finally sent the mutated fragment to sequencing, confirmed that the mutation was correct and then ligated into the enzymatically cleaved pGADT7 by infusion technology, and then performed yeast double hybridization. (Figure: sequencing results, ligated electrophoresis, yeast double hybridization)

### Analysis and Learning

Finally, we found that the results of yeast double hybridization were not obvious, and we suspected that there was a problem in the vector sequence construction, we added the nls sequence of yeast system RGRGRGRGRGRGRGRGGYRGRARGFAPY\* to the sequence of point mutation in the subsequent experiments to conduct another experiment

## Random Peptide Library

### Design

In order to find the peptide with strong interaction with spike, we constructed a platform that can generate 20 amino acids randomly. And a series of peptides are generated by an exhaustively exampled method. This approach can generate a database of large capacity. We perform yeast two-hybrid interaction screening for the peptides in this database, and we can get a series of peptides that can interact strongly with the target protein.We then compared the similarities on the ACE2 sequences of these peptides and other species by bioinformatics approach, wanting to find potential viral hosts.

By reading the papers, we found that the library capacity of the random peptide library would be relatively large, so we prepared to construct the random peptide library fragment to pGADT7GW vector by gateway method, so we first constructed the random peptide library fragment containing attb sequence.

![](https://static.igem.wiki/teams/4253/wiki/enginneer-success/14.jpg)

Figure2. Random peptide library sequences

![](https://static.igem.wiki/teams/4253/wiki/enginneer-success/15.jpg)

Figure3. pDONR207-random peptide vector and pGADT7 GW-random peptide vector

### Construct

The random DNA sequences encoding the 20 peptides were synthesized by Shanghai Biological Co. (N represents A, T, G, and C, K represents G and T.) Twenty short peptides contain attb1 and attb2 sites in the upper and lower sides of the sequence, respectively To terminate the translation of the random 20 peptide, the TAA stop codon was designed after the random 20 short peptide. In order to amplify a random DNA fragment of 20 short peptides, we designed two additional primers for the random DNA fragment (synthesized by Shanghai Biological Co.) After obtaining the sequence, we performed PCR amplification immediately, and then recovered the sequence and transformed it into pDNOER 207 vector. By using gateway bp reaction, and finally transformed into pGADT7-GW vector by LR reaction

![](https://static.igem.wiki/teams/4253/wiki/enginneer-success/16.jpg)

Figure4. random peptide Build process：bd-bait successfully transferred into yeast AH109 without self-activation

### Testing

We first transformed the spike protein sequence into the AH109 strain and tested it for self-activation. After confirming the absence of self-activation, we co-transformed this AH109 strain with the Y187 strain containing the secondary library. Finally, Cotransformants are plated on SD/-His/-Leu/-Trp medium. After this screening, we obtained 36 positive strains, in order to analyze the protein sequence characteristics we obtained, we performed colony PCR and detected its sequence information by sequencing analysis.

![](https://static.igem.wiki/teams/4253/wiki/enginneer-success/3.jpg)

Figure5. Random peptide library transformation results confirm DK2 as the strongest binding short peptide with covid-19

### Analysis and Learning

These results allow us to conclude that the construction of a random peptide library will allow us to quickly screen for interacting proteins of relevance to them for rapid detection when preventing the arrival of the next outbreak

## PmrCAB Two-component system

### Design

The PmrCAB system was a system to sense Fe(III) in the outside world for Salmonella originally, where we envisioned replacing the iron-binding motif of PmrB (amino acids 34 - 64) with the sequence of a random peptide library we have obtained to be highlighted in green. The relationship between viral concentration and final fluorescence signal intensity was then established using different concentrations of the previously described spike protein.

![](https://static.igem.wiki/teams/4253/wiki/design/7.jpg)

Figure6. Design and construction of a lanthanide-responsive system based on the PmrA/PmrB two-component system

### Construct

We have changed the specific sequence of AffiPmrB in the strain provided by Tsinghua, replacing the binding motif with the sequence of our random peptide library.

### Testing

To establish the relationship between final virus concentration and fluorescence intensity, characterization experiments were performed as follows:

When the bacteria were grown to the appropriate stage, IPTG was added to induce the expression of PmrB and PmrA. A certain gradient of spiked protein (after optimization), corresponding to three types of coronaviruses, was taken to stimulate the sensor PmrB to turn on a series of responses afterwards. Each concentration corresponds to one fluorescence concentration.

We also used a modeling approach to establish a theoretical relationship between virus concentration and fluorescence intensity.

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/2.png)

Figure7:Relationship between spike protein concentration and fluorescence intensity(left：spike induction results under fluorescence microscope. The engineering bacteria was induced with 1.0 μg/mL~3μg/ml spike(after codon optimization). Photos were taken under fluorescence microscope.right：the theoretical relationship between spike protein concentration and fluorescence intensity was made by mathematical modeling method based on the principle of two-component system)

### Analysis and Learning

We compare the experimental results with the modeling results. It was found that the general trends were in accordance with each other. However, the specific amounts differed considerably, which may be due to the wrong prediction of our peptide library and spike binding capacity parameters, and our modeling did not take into account factors such as bacteriophage concentration.

However, we can roughly conclude that the detection limit of our cell sensor is about 1 μg/ml, which is not sufficient for virus detection in wastewater. So we thought we needed to concentrate the virus before detection.

## Suicide part

### Design

To prevent our bacteria from overflowing into the environment. We designed the circuit for light-coupled suicide, which needs to be used together with our subsequent circuit, which requires us to precisely control the experimental conditions to determine the time of bacterial death after the light-controlled conditions are turned on.

To this end, we figured out the mechanism of bacteriocin cleavage in E. coli by modeling and wanted to obtain less toxic proteins by individual amino acid transformations. However, after comparing several options, we chose the original one and a blue light intensity of 150 HZ.

![](https://static.igem.wiki/teams/4253/wiki/enginneer-success/6.png)

### Construct

We changed the mRFP part of the previous plasmid to BBa K117000 element and cultured it.

### Testing

We incubated the bacteria with blue light irradiation after coating the plates. And another batch of bacteria was cultured under the same conditions by avoiding light, and finally these two batches of bacteria were cultured under the same conditions.

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/5.png)

Figure8.The three above：Darkness nurtures The next three：bu lights 100μmol/m2/sCulture for 20 hours

### Analysis and Learning

We can find that the growth of bacteria is significantly inhibited under blue light, while bacteria not irradiated with blue light have no effect. This shows the success of our light-coupled suicide circuit.

light-coupled suicide circuit.
We also found that in the already grown bacteria did not die by lysis in a large area under the condition of blue light irradiation, but we found that blue light did not significantly inhibit the growth of E. coli in our project last year. There may be a mechanism that we are not aware of.

## the Escape Part

### Design

To be able to concentrate virus in wastewater, we constructed an engineered bacterium capable of "transporting" the virus. We used light control elements to regulate the expression of a flagellar protein kinase. This kinase dephosphorylates CheY, causing the flagellum to rotate counterclockwise. This change causes E. coli to move forward at a faster rate. When CheZ is not expressed in the exogenous gene, the flagellin in E. coli will rotate clockwise, causing the E. coli to "roll" in the same place.We want to build the "light avoidance" of E. coli, when there is blue light exposure, E. coli will move at high speed to find a "way out", otherwise it will make too much virulence protein expression that would cause E. coli lysis and death if it is exposed to blue light for a long time. Thus, E. coli can only "carry" the adherent virus "escape" to the area without blue light.

![](https://static.igem.wiki/teams/4253/wiki/design/4.jpg)

Figure9.The mechanism by which E. coli "transport" viruses

### Testing

We implanted the "escape system" in the above constructed E. coli with the suicide gene and implanted pigment proteins to indicate the location of the E. coli. Then we constructed a part of the blue light-free area with tin foil. Then we incubated with blue light.

### Analysis and Learning

The final biological image has blurred border between the region without blue light and the region with blue light, probably due to the fact that we did not knock out CheZ in E. coli, which made some of the E. coli in the region without blue light escape back to the region with blue light.

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# RESULT

[Identification of Artificial SPIKE-Interacted Small Peptides](#p-1)
[Validation of a suicide system](#p-2)
[Validation of two-component systems](#p-3)
[Validation of the escape system](#p-4)
[SOE PCR](#p-5)

## Identification of Artificial SPIKE-Interacted Small Peptides

### 1. Construction of random library

The random fragment (iGEM\_Fragment-1) was synthesized, and the double-strand sequence were obtained by PCR amplification with fragment amplifying primers (Random\_Primer1, Random\_Primer2). The obtained PCR products were constructed into entry vector pDONR207 by Gateway BP reaction, resulting in the primary library pONR207-random peptide (Figure 1). The pDONR207-random peptide plasmid library was subsequently constructed into the destination vector pGADT7-GW through Gateway LR reaction (Figure 2), resulting in a yeast two-hybrid AD plasmid library. The secondary plasmid library (pGADT7 GW-random peptide) was then transferred into the yeast Y187 strain, resulting in a Y187 AD-tagged random-peptide library (Figure 3).

1. Adaptor information for the Gateway system

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/16.png)

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/14.png)

Figure 1. The pDONR207 was used for the construction of the Entry library. The cyan bar shows the DNA sequences coding the random peptide library.

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/15.png)

Figure 2. The pGADT7 GW was used for the construction of the secondary library. The gray bar shows the DNA sequences coding the random peptide library.

![](https://static.igem.wiki/teams/4253/wiki/result/03.jpg)

Figure 3. The colonies of AD-random Libray in AH109 strain.

### 2. The characteraztion of the Yeast two hybrid Library

The 150-bp insert fragments were amplified using primers CCAGATTACGCTCATATGACAAGTTTGTAC and TCATCTGCAGCTCGAGACCACTTTGTACAA. The constructed libraries were sequenced using the Illumina Novaseq 6000 platform to generate raw data, following the manufacturer’s instructions by the company of ANNOROAD Biotech. Co., Ltd. (China). After quality control, about 34.5 GB of clean data were generated. According to the design principles (TCGTCGGGGACAACTTTGTACAAAAAAGTTGGAACC-(NNK)20-TAAGACCCAACTTTCTTGTACAAAGTTGTGCGGCCGCC), 60bp random sequences were extracted from the clean data and translated into amino acid sequences using standard genetic codons. The translation was stopped from the first stop codon, and peptides less than five aa in length after translation were removed. Finally, we obtained 68,190,232 peptides in total and 3,359,176 peptides after removing redundancy. (table1)

### Table 1: Statistics of sequencing results

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/9.png)

We carried out saturation analyses and found that about 35Gb data was far from covering all the kinds of peptides in our random peptide library (Figure 4A), indicating that our random peptide library has a high diversity and a broad potential for protein interaction investigations and applications.

![](https://static.igem.wiki/teams/4253/wiki/result/04.jpg)

Figure 4. statistics of the sequencing data about our random peptide library. (A) Statistics of the peptide number of different lengths; (B) Saturation analyses according to randomly sampling from the sequencing data. The abscissa represents the proportion of random sampling including 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, and the ordinate represents the number of peptides detected corresponding to the percentage of sampled sequencing data.

### 3. The screening of SPIKE-RBD binding peptides

AH109 strain was transformed with pGWBD\_Spkie-RBD plasmid to obtain the bait construction of yeast two-hybrid. Through the yeast two-hybrid mating system (Figure 5A), 36 positive colonies were finally obtained (Figure 5B). After amplification with AD-REV and AD-FWD primers, 35 PCR fragments were obtained, and then 34 random peptide library sequences were obtained by sanger sequencing (Table 1).

![](https://static.igem.wiki/teams/4253/wiki/result/05.jpg)

Figure 5. The screening of SPIKE-RBD binding peptides through Y2h. (A) The microscopy of the mating process of AH109 and Y187. (B) The positive clonies of mating yeast gown on the –Trp/-Leu/-His/-Ade SD medium.

#### Table 1.The aa sequences of 34 SPIKE-RBD binding peptides

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/4.png)

We aim to find the strongest short peptide sequence that can interact with the RBD structural domain of Spike, the 34 sequences were aligned, and the conserved domains were obtained (Figure 6). Moreover, the consensus sequence WGPMLWRAFDRAMSAGLGGW was obtained, which is the short peptide that we think has strong interaction with the Spkie RBD domain.

![](https://static.igem.wiki/teams/4253/wiki/result/06.jpg)

Figure 6. Multiple sequence alignment of the 34 peptides interacted with Spike, the quality score is calculated for each site in the alignment by summing, and a consensus sequence was generated according to the alignment analysis.

## Validation of a suicide system

In order to adapt our toxic proteins to the escape system, we made some modifications to the lytic protein.The best concentration is achieved by reducing the toxicity of the toxic protein.We have modified some amino acids so that the bacteria do not die too quickly when expressing the protein.In the end, we chose one option and transferred the gene into a JLU-China's light-controlled parts. Finally, we grow this bacteria under the blue light condition.

![](https://static.igem.wiki/teams/4253/wiki/result/7.jpg)

Figure 7.the suicide plasmid

The final results showed significant growth suppression within 10 to 20 hours after the insertion of the suicide gene, as we expected

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/5.png)

Figure5:The three above：Darkness nurtures The next three：bu lights 100μmol/m2/sCulture for 20 hours

## Validation of two-component systems

To determine the detection limit and linear range of our cellular sensors. We established the relationship between the final fluorescence intensity and spike protein concentration by modeling.After obtaining our peptide library sequence, we transfered the sequence into the sensing unit of the PmrB (thanks to the strain provided by Tsinghua) , followed by the Spike protein after the optimized sequence adopted by the yeast doublet previously
We did a series of gradient experiments, and the final results were similar to the modeling results.However, the detection limit was too high (that is, the sensitivity of the two-component system to the optimized spike protein was too low) . So we wanted to use peptides that could"Capture" Spike's properties to concentrate the virus.

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/2.png)

Figure9:Relationship between spike protein concentration and fluorescence intensity(left：spike induction results under fluorescence microscope. The engineering bacteria was induced with 1.0 μg/mL~3μg/mL spike(after codon optimization). Photos were taken under fluorescence microscope.right：the theoretical relationship between spike protein concentration and fluorescence intensity was made by mathematical modeling method based on the principle of two-component system)

## Validation of the escape system

To enrich our virus, we wanted to use the peptides to“Catch” the virus by binding specifically to the spike.But after catching the virus, we also need E. coli to be able to move in a given direction.For ease of use, we chose blue light as our trigger mechanism.

When irradiated with blue light, E. coli can move in a straight line at high speed because of the large amount of expression of this gene, but not in the region irradiated with blue light. E. coli rolls around because of the knock-out of the gene.
After some time, E. coli will accumulate in areas without blue light. This is the“Light-repellent” we construct for E. coli. Take advantage of this property,E. coli then“Carries”the protein to areas where there is no blue light.

## SOE PCR

First, we simulated the structure of HACE2 based on the RBD with SARS-CoV-2 using UCSF Chimera and predicted the site of enhanced HACE2 For these sites, we designed corresponding primers to modify and synthesize the PCR productsWe recovered the pcr reaction solution and sequenced it to make sure that all the mutations were successful, then we recovered the fragments and constructed the vectors by infusion ligation. Finally we used the constructed vector to perform yeast two-hybrid, but unfortunately we found that the vector we designed did not show their ability to interact with each other in this way after performing yeast two-hybrid, so we communicated with the PI and wondered if the nls had not been added and thus no yeast two-hybrid did not show the results it should, so we modified the experiment again and gave our mutant sequence with the yeast nls fragment RGRGRGRGRGRGRGRGGYRGRARGFAPY\*(Table3)

#### Table 3.ACE2 point mutation predicted enhancement sites

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Number | Spike binding sites | ACE2 | Distance change before and after mutation | Binding energy | Predictions |
| 1 | SARS-CoV-2 | hACE2 | Original sequence | -14.38 | / |
| 2 | Q493 | l\_21\_V | 6.541->6.506 | -14.15 | Stronger |
| 3 | F486 | K\_26\_R | 5.132->3.8007 | -14.38 | Stronger |
| 4 | F486 | T\_27\_A | 9.886->9.387 | -14.39 | Stronger |
| 5 | L455 | K\_31\_R | 13.985->11.889 | -14.37 | stronger |
| 6 | F486 | N\_33\_I | 8.529->8.309 | -14.37 | Stronger |
| 7 | F486 | H\_34\_R | 15.184->12.349 | -14.36 | Stronger |
| 8 | F486 | E\_37\_K | 15.175->10.415 | -14.35 | Stronger |
| 9 | F486 | T\_92\_I | 7.047->7.0247 | -14.38 | Stronger |

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/10.png)

Figure10: Figure E and F show the atomic spacing before and after the mutation of the F486 site of Spike with the site 34 (H) of ACE2.Figure G and H show the atomic spacing before and after the mutation of the L455 site of Spike with the site 31 (K) of ACE2.

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/11.png)

Figure11:Figure I and Figure J show theatomic distance between theQ493 site of Spike and the 21（I )site of ACE2 before and after mutation.Figure K and Figure L show theatomic distance between the F486site of Spike and the 27 (T) site ofACE2 before and after mutation.

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/12.png)

Figure12: Figure M and Figure N show theatomic distance between the F486site of Spike and the 26 (K) site ofACE2 before and after mutation.Figure 0 and Figure P show theatomic distance between the L455site of Spike and the 92 (T) site ofACE2 before and after mutation.

![](https://static.igem.wiki/teams/4253/wiki/niuma/sibo/13.png)

Figure Qand Figure R show theatomic distance between theF486 site of Spike and the 33 (N)site of ACE2 before and after mutation.Figure S and Figure T show theatomic distance between theL455 site of Spike and the 37 (E)site of ACE2 before and after mutation.

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