iGEM 2024 — Team squirrel-shanghai

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
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It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.wiki/teams/5131/notebook/lab-notebook-squirrel-sh-v1.pdf)

### Or Download it!

[Lab Notebook](https://static.igem.wiki/teams/5131/notebook/lab-notebook-squirrel-sh-v1.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
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[Overview](#overview)
[Round 1 Expression and purification of nsp5](#round1)

[Design](#design-1)
[Build](#build-1)
[Test](#test-1)
[Learn](#learn-1)
[Round 2 Expression, purification, and enzymatic activity characterization of nsp5\_native](#round2)

[Design](#design-2)
[Build](#build-2)
[Test](#test-2)
[Learn](#learn-2)
[Round 3 Development of an in vivo nsp5 inhibitor screening platform based on FlipGFP](#round3)

[Design](#design-3)
[Build](#build-3)
[Test](#test-3)
[Learn](#learn-3)
[Round 4 Rational design of nsp5](#round4)

[Design](#design-4)
[Build](#build-4)
[Test](#test-4)
[Learn](#learn-4)

## Overview

We follow the design-build-test-learn(DBTL) cycle to progressively advance our project. In summary, our project can be divided into the following four cycles: expression and purification of nsp5, expression, purification, and enzymatic activity characterization of nsp5 with natural N- and C-termini, development of an in vivo inhibitor screening platform based on FlipGFP, and rational design of nsp5 to enhance its enzymatic activity.

## Round 1 Expression and purification of nsp5

#### Design

Due to our ultimate goal of achieving in vivo screening of nsp5 inhibitors, our initial goal is to successfully express functionally active nsp5 protein in an *E. coli* host by optimizing the expression conditions and using an appropriate purification strategy. To achieve this goal, we first designed an expression vector for the production of SARS-CoV-2 nsp5. To ensure soluble expression of the protein, we selected the pGEX-6P-1 vector, which includes a GST tag and an HRV 3C protease cleavage site. For purification, we fused a 6\*His tag to the C-terminus of the nsp5 sequence while retaining the HRV 3C protease cleavage site for subsequent removal of the tag(Figure 1).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-1.webp",
caption="Figure 1. Sequence design for GST-nsp5-His.",
width="70%"
) }}

#### Build

We first successfully amplified the vector backbone and the nsp5-6\*His tag separately using PCR (Figure 2B). Subsequently, we constructed the pGEX-GST-nsp5-His through homologous recombination. The sequencing results confirmed the correct construction of our vector (Figure 2C).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-13.webp",
caption="Figure 2. A) Vector design of pGEX-GST-nsp5-His. B) PCR amplification of nsp5 and pGEX-6P-1. C) Sequencing validation of nsp5.",
width="70%"
) }}

#### Test

We expressed the protein in *E. coli* BL21 and purified it using Ni-NTA affinity chromatography. First, *E. coli* BL21 cells transformed with the pGEX-GST-nsp5-His plasmid were cultured at 37°C until the OD reached ~0.6. Then, protein expression was induced by adding IPTG to a final concentration of 0.2 mM. After 16 hours of induction, the cells were lysed, and the supernatant was collected by high-speed centrifugation (13,000 xg). The supernatant was passed through a Ni-NTA affinity column to allow the target protein to bind to the Ni-NTA beads. Next, HRV 3C protease was added to remove the excess amino acids from the N- and C-termini of the target protein, separating it from the Ni-NTA beads. Finally, the purified SARS-CoV-2 nsp5 was eluted and concentrated for further analysis. SDS-PAGE analysis showed that the purified and cleaved protein had high purity. However, the band appeared around 70 kDa(Figure 3), which differs from the expected molecular weight of nsp5 (33.8 kDa).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-15.png",
caption="Figure 3. Purification of nsp5. Lane 1-8: marker, control group, supernatant after centrifugation, nickel beads before digested, flow-through buffer, eluted buffer, digested nickel beads, purified protein.",
width="70%"
) }}

#### Learn

During this cycle, we found that the molecular weight of the purified protein differed from the expected value, suggesting that the protein obtained through this expression and purification method might be in a non-native state, implying incorrect folding or impaired activity.

After carefully reviewing the clone, we discovered that the theoretical molecular weight of the GST tag + nsp5 was approximately 60 kDa, which was close to the observed band position. This indicated that the larger-than-expected molecular weight was likely due to the incomplete removal of the GST tag. Since there were no redundant amino acids between the HRV 3C protease cleavage site and the nsp5-GST tag, we hypothesized that steric hindrance at the cleavage site was preventing HRV 3C protease from accessing the site and cleaving the GST tag. In contrast, the C-terminal His tag did not have this issue, and our protein structure prediction modeling(Figure 4) confirmed this observation (see [Model](https://2024.igem.wiki/squirrel-shanghai/model) for details). Therefore, according to this vector design, we obtained nsp5 with a native C-terminus but a redundant GST tag at the N-terminus.

To address this issue, we proposed two solutions. One approach was to introduce a flexible GGGS linker upstream of the nsp5 sequence to expose the cleavage site. However, this would still leave redundant amino acids at the N-terminus of nsp5. The other approach was inspired by the SARS-CoV-2 lifecycle[1], where nsp5 must undergo self-cleavage at the N-terminus to release itself from the polyprotein during normal viral processing. Therefore, we considered introducing the self-cleavage sequence of nsp5 at its N-terminus (referred to as nsp5\_native) to allow the N-terminal GST tag to be self-cleaved after protein expression, resulting in SARS-CoV-2 nsp5 with both native N- and C-termini.

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-4.webp",
caption="Figure 4. Predicted structure of GST-nsp5, where nsp5 is shown in orange, the GST tag in green, the HRV 3C protease recognition region in cyan, and the amino acid residues at the cleavage site in yellow.",
width="70%"
) }}

## Round 2 Expression, purification, and enzymatic activity characterization of nsp5\_native

#### Design

Based on previous experience, we introduced four amino acids (AVLQ) at the N-terminus of nsp5 to facilitate its self-cleavage from the GST tag. The rest of the sequence remained consistent with the pGEX-GST-nsp5-His construct(Figure 5). This new construct is designated as pGEX-GST-nsp5\_native-His.

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-5.webp",
caption="Figure 5. Sequence design for GST-nsp5\_native-His.",
width="70%"
) }}

#### Build

Following the same vector construction method as in round 1, we successfully constructed pGEX-GST-nsp5\_native-His and confirmed its accuracy through sequencing analysis(Figure 6B).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-6.webp",
caption="Figure 6. A) Vector design of pGEX-GST-nsp5\_native-His. B) PCR amplification of nsp5\_native and pGEX-6P-1. C) Sequencing validation of nsp5\_native.",
width="70%"
) }}

#### Test

Following the same purification steps as in round 1, we successfully purified nsp5\_native. SDS-PAGE indicated that nsp5\_native had high purity and a molecular weight consistent with expectations. We subsequently measured the efficiency of nsp5\_native in cleaving a fluorescent substrate using FRET, and characterized the *kcat*/*Km* of nsp5\_native as 27,691 s⁻¹M⁻¹(Figure 7) based on the Michaelis-Menten equation(see [Model](https://2024.igem.wiki/squirrel-shanghai/model) for details). From the SDS-PAGE and FRET results, we concluded that nsp5\_native possesses the natural N- and C-termini of SARS-CoV-2 nsp5.

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-7.jpg",
caption="Figure 7. Purification of nsp5\_native. Lane 1-3: marker, digested nickel beads, purified protein(left panel). Kinetic model of nsp5 enzyme activity(right panel).",
width="70%"
) }}

#### Learn

After two rounds of the DBTL cycle, we have confirmed that it is possible to synthesize SARS-CoV-2 nsp5 with a native N-terminus and C-terminus, as well as correct functionality, in *E. coli* BL21 using the existing expression strategy. This lays a solid foundation for the subsequent construction of the in vivo inhibitor screening platform. Furthermore, we noticed that nsp5 exhibits extremely high activity when cleaving substrates, prompting us to consider other potential applications for nsp5. The protein purification steps indicate that the removal of the recombinant tag is a key part of the entire purification process. nsp5's enzymatic activity is nearly 30 times higher than that of the commonly used TEV protease and HRV 3C protease for tag removal, suggesting significant potential for nsp5 in this application. Therefore, introducing some mutations to enhance nsp5's enzymatic activity could make it a more effective tool enzyme.

## Round 3 Development of an in vivo nsp5 inhibitor screening platform based on FlipGFP

#### Design

The in vivo screening platform has advantages such as avoiding the cumbersome process of protein purification, being closer to physiological conditions, and reflecting drug toxicity. In this round of the project, we primarily aimed to design an in vivo nsp5 inhibitor screening platform. To achieve this, we selected FlipGFP[2], a fluorescent protein that responds to proteases, as our reporter gene. Under ideal conditions, FlipGFP does not emit fluorescence when expressed alone, but it fluoresces upon recognition and cleavage by a protease (see [Description](https://2024.igem.wiki/squirrel-shanghai/description) for details). Therefore, the intensity of fluorescence can indicate the activity of the protease.

To ensure the correct expression of FlipGFP, we used the pRSF-Duet1 and separately inserted the first nine β-strands of FlipGFP and the engineered 10-11 β-strands into two different ORFs. This vector is referred to as pRSF-FlipGFP\_nsp5(10-11)-FlipGFP(1-9). To facilitate inhibitor screening, we designed a genetic circuit with the following functions(Figure 8): when only FlipGFP is present, the entire system does not emit fluorescence; when both nsp5 and FlipGFP are present, nsp5 cleaves FlipGFP, resulting in fluorescence; and when nsp5 is inhibited, the cleavage efficiency of nsp5 on FlipGFP decreases, leading to reduced or absent fluorescence.

{{ figure(
image="https://static.igem.wiki/teams/5131/design/design-5.png",
caption="Figure 8. Genetic circuit of nsp5 activated FlipGFP",
width="70%"
) }}

#### Build

Following the same vector construction method as in round 1, we successfully constructed pRSF-FlipGFP\_nsp5(10-11)-FlipGFP(1-9) and confirmed its accuracy through sequencing (Figure 9B).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-8.webp",
caption="Figure 9. A) Vector design of pRSF-FlipGFP\_nsp5(10-11)-FlipGFP(1-9). B) Sequencing validation of FlipGFP\_nsp5(10-11)-FlipGFP(1-9).",
width="50%"
) }}

#### Test

We separately transformed *E. coli* BL21 with FlipGFP alone and co-transformed BL21 with FlipGFP and nsp5, then plated them on LB plates containing IPTG at a final concentration of approximately 0.2 mM. The results showed that colonies transformed with FlipGFP alone produced almost no fluorescence, while colonies co-transformed with FlipGFP and nsp5 emitted significant fluorescence. This demonstrates that we successfully constructed a FlipGFP system that can be activated by nsp5 to emit fluorescence (Figure 10).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-9.webp",
caption="Figure 10. Activation of FlipGFP by nsp5.",
width="70%"
) }}

#### Learn

We successfully constructed a FlipGFP system that can be activated by nsp5, confirming the great potential of our platform for inhibitor screening. However, for practical applications in the future, there are several points for improvement:

- Introduce known nsp5 inhibitors (such as disulfiram, ebselen, nirmatrelvir, etc.) into the existing system to assess the platform's sensitivity to inhibitors.
- Use liquid LB culture of the co-transformed strains and measure the changes in fluorescence intensity with a plate reader to establish a more comprehensive inhibitor screening model.
- Transfer the entire system to an eukaryotic cell system to make the screening environment closer to the actual usage scenario of the inhibitors.

Since FlipGFP can respond to other proteases with only minor modifications to the recognition sequence, this indicates that our designed platform also possesses good scalability, allowing it to be adapted for screening other protease inhibitors with minimal changes. Considering the importance of proteases in the lifecycle of all positive-strand RNA viruses, we believe this platform can be quickly utilized for screening new inhibitors in the event of future outbreaks of other positive-strand RNA viruses. Compared to methods that require protein purification for inhibitor screening, our platform is more flexible and efficient.

## Round 4 Rational design of nsp5

#### Design

TEV protease[3] and HRV 3C protease[4] are commonly used tool enzymes for the removal of recombinant tags. Compared to them, nsp5 has higher enzymatic activity, suggesting that nsp5 may be better suited for this purpose. In this cycle, we aimed to enhance the enzymatic activity of nsp5 through rational design to improve its efficacy in removing recombinant tags. To achieve this, we first predicted the structure of the nsp5-substrate complex. We hypothesized that increasing the interaction strength between the substrate and nsp5 could enhance enzymatic efficiency. Consequently, we designed a mutation, T21I, that might improve enzyme activity. Structural predictions indicated that this mutation increases the interaction between the substrate and the 21st amino acid of nsp5, making it a potential variant with enhanced activity (Figure 11). We used the same vector design as in the second cycle, naming the vector pGEX-GST-nsp5\_T21I-His.

![](https://static.igem.wiki/teams/5131/engineering/engineering-success-10.webp)

Figure 11. Interaction between wild-type nsp5 and nsp5-T21I with the R10substrate, with an interaction distance threshold of 4 Å and the interaction indicated by yellow dashed lines. Wild-type nsp5(left panel) is shown in green, while nsp5-T21I(right panel) is shown in cyan.

#### Build

In order to introduce the mutation at the 21st amino acid, we used the point mutation method to construct the vector . We designed a pair of primers at the corresponding position in the DNA sequence and replaced the codon corresponding to T with the codon corresponding to I in the DNA sequence by reverse PCR. We then transformed this linear sequence into *E. coli* DH5α and used its own repair mechanism to obtain the vector pGEX-GST-nsp5\_T21I-His. We confirmed its accuracy through sequencing analysis(Figure 12B).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-11.webp",
caption="Figure 12. A) Vector design of pGEX-GST-nsp5\_T21I-His. B) Sequencing validation of nsp5-T21I.",
width="50%"
) }}

#### Test

Following the same purification steps as in round 1, we successfully purified nsp5-T21I. SDS-PAGE indicated that nsp5-T21I had high purity and a molecular weight consistent with expectations(Figure 13).This suggests that the nsp5-T21I has native N- and C-termini.

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-17.png",
caption="Figure 13. Purification of nsp5-T21I. Lane 1-2: marker, purified protein.",
width="30%"
) }}

We subsequently measured the enzymatic activity of nsp5-T21I using FRET, and characterized the *kcat*/*Km* of nsp5-T21I as 35,069 s⁻¹M⁻¹ based on the Michaelis-Menten equation(see [Model](https://2024.igem.wiki/squirrel-shanghai/moddel) for details). From FRET results, we confirmed that the nsp5 T21I variant indeed possesses higher enzymatic activity compared to the wild-type nsp5(Figure 14).

{{ figure(
image="https://static.igem.wiki/teams/5131/engineering/engineering-success-12.webp",
caption="Figure 14. Kinetic model of nsp5-T21I(left panel). Reaction rates comparsion between WT nsp5 and nsp5-T21I(right panel).",
width="70%"
) }}

#### Learn

We successfully utilized rational design to enhance the enzymatic activity of nsp5, making it better suited for the removal of recombinant tags and demonstrating its potential as a tool enzyme. Our results confirm that increasing the affinity between nsp5 and its substrate contributes to enhanced activity, providing important references for further design and optimization of nsp5. In the future, we can explore additional mutations at various sites and combinations to further boost the enzymatic activity of nsp5, ultimately aiming to develop it into a universal tool enzyme for recombinant tag removal.

## References

1. Yang H, Rao Z. Structural biology of SARS-CoV-2 and implications for therapeutic development. Nat Rev Microbiol. 2021 Nov;19(11):685-700. doi: 10.1038/s41579-021-00630-8. Epub 2021 Sep 17. PMID: 34535791; PMCID: PMC8447893.
2. Zhang Q, Schepis A, Huang H, Yang J, Ma W, Torra J, Zhang SQ, Yang L, Wu H, Nonell S, Dong Z, Kornberg TB, Coughlin SR, Shu X. Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals. J Am Chem Soc. 2019 Mar 20;141(11):4526-4530. doi: 10.1021/jacs.8b13042. Epub 2019 Mar 6. PMID: 30821975; PMCID: PMC6486793.
3. Parks TD, Howard ED, Wolpert TJ, Arp DJ, Dougherty WG. Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form. Virology. 1995 Jun 20;210(1):194-201. doi: 10.1006/viro.1995.1331. PMID: 7793070.
4. Wang QM, Johnson RB. Activation of human rhinovirus-14 3C protease. Virology. 2001 Feb 1;280(1):80-6. doi: 10.1006/viro.2000.0760. PMID: 11162821.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

[Reagent List](#reagent)
[Laboratory Protocol](#laboratory)

[1. Plasmid extraction](#laboratory-1)
[2. Digestion vectors](#laboratory-2)
[3. PCR](#laboratory-3)
[4. Agarose Gel Electrophoresis & Gel Extraction](#laboratory-4)
[5. Homologous recombination ligation transformation](#laboratory-5)
[6. Protein expression purification](#laboratory-6)
[7. SDS-PAGE](#laboratory-7)
[8. Co-transformation and enzyme activity](#laboratory-8)
[9. Inhibitors impact](#laboratory-9)

## Reagent List

1. 1×TAE
2. 1% agarose powder
3. BL21 competent cell
4. DH5α competent cell
5. Ampicillin
6. kanamycin
7. LB Broth (25 g/L)
8. LB Broth Agar (40 g/L)
9. HRV-3C Protease
10. 1 M & 0.1 M IPTG
11. Purification Buffer (500 ml): 20 mM Tris-HCl pH 8.0, 300 mM NaCl
12. Wash Buffer (500 ml): 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM Imidazole
13. Balance Liquid BL
14. Buffer P1: 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM Glucose
15. Buffer P2: 250 mM NaOH, 1% SDS
16. Buffer P3: 3 M potassium acetate, 5 M acetate acid
17. Buffer PW (wash buffer): 10 mM Tris-HCl (pH 7.5), 80% ethanol
18. Buffer EB (elution buffer): 10 mM Tris-HCl (pH 7.5)
19. Coomassie Brilliant Blue G250
20. 1×Tris-Glycine-SDS electrophoresis running buffer
21. DTT
22. 2×Loading Buffer
23. 2×Phanta Flash Master Mix
24. Ensitrelvir
25. Disulfiram
26. Dpnl
27. Color prestrained protein marker (10-180 kDa)
28. Feto Protein straining buffer
29. MCA-AVLQS GFRK (DnP) K 9MG 95
30. ClonExpress II One Step Cloning Kit

## Laboratory Protocol

#### 1. Plasmid extraction

* Column equilibration：place the adsorption column CP3 into the collecting pipe and add 500 μl equilibrium
  liquid BL. Centrifuge the liquid at 12000 rpm for 1 min. Pour out the liquid in the pipe.
* Take 1.5 ml bacteria solution which has been cultured overnight and add into centrifuge tube. Centrifuge it at
  12000 rpm for 1min then pour out the supernate. Repeat this step until all the bacteria solution is used.
* Add 250 μl P1 solution and suspend the bacteria in the solution by using the vortex oscillator (The P1
  solution mainly consists glucose, EDTA and Tris C. Glucose increases the viscosity of the solution, reduces the
  mechanical shear force, and prevents plasmid DNA damage;EDTA chelates divalent metal ions, such as Mg²⁺, to
  prevent the degradation of plasmids by DNA enzymes; Tris CI maintains the optimal pH range of the lysate to
  ensure the activity of lysozyme.)
* Add 250 μl P2 solution and turn it upside down 6-8 times soft to make the bacteria fully cleavage. (The P2
  solution consists SDS and NaOH.SDS binds to proteins and denatures them, and NaOH (pH>12) breaks hydrogen bonds
  and denatures the DNA molecule. This step focuses on disrupting the cell wall and releasing the DNA.)
* Add 350 μl P3 solution and turn it upside down for 6-8 times immediately.Now there is white flocculent
  precipitate.Then centrifuge it at 12000rpm for 10min. (The P3 solution contains KAc and HAc which form a
  high-salt solution with a pH of 5.5. It can neutralize the alkalinity of P2 solution and restore chromosomal DNA
  and plasmid DNA. K⁺ ions form low solubility salts with SDS and form precipitates with proteins for removal. The
  high-salt environment helps the formation of macromolecular precipitates, Thus isolating plasmid DNA.)
* Remove the supernate into the adsorption column CP3 by the pipette.(Do not remove the precipitate into the
  column).Then centrifuge it at 12000 rpm for 60 s.Then pour out the liquid in the collecting tube.
* Add 600μl PW bleach wash solution which should be added absolute ethyl alcohol in advance.Then centrifuge it
  at 12000 rpm for 60 s and pour out the liquid in the collecting tube.
* Repeat the 7th step
* Put the adsorption pipe into the collecting tube and centrifuge it at 12000 rpm for 2 min in order to wipe off
  the remaining bleach wash solution.
* Put the adsorption pipe into clean centrifuge tube and add 50 μl elution buffer.Place at the indoor
  temperature for 2 min.Centrifuge it at 12000 rpm for 2 min. Now the plasmid solution is in the centrifuge tube.

#### 2. Digestion vectors

The plasmid was digested with a specific endonuclease at a temperature of 37°C and a digestion time of 1 h.

#### 3. PCR

The system of PCR included: nsp-5 template plasmid, 2×Phanta Flash Master Mix, forward primer reverse primer ,
ddH2O.

PCR procedure：

1. 98℃-30 s
2. 98℃-10 s
3. 60℃-5 s
4. 72℃-30-180 s（extension rate 30s/kb）
5. 72℃-10 min
6. 25℃-5 min

Steps ②-③-④ repeat 30-35 times

#### 4. Agarose Gel Electrophoresis & Gel Extraction

* Run PCR products in the 1% agarose gel, lets the product undergo 120V electrophoresis for about 30-40 min.
  After AGE is completed, analyze the position of the product in the agarose gel, should be about 6000 bp. After
  comfirm the target sequences, the gel recovery of the nsp-5 begin.
* Place the single target DNA band cut from the agarose gel into a clean centrifuge tube and weigh
* Add an equal volume of solution PN to the gel in a 50℃ water bath
* Add the solution to adsorption column CA2 and place it for 2 min at room temperature
* Centrifuge (12000 rpm, 60 s)
* Wash with 600 μl PW, centrifuge (12000 rpm, 60 s) , pour the discard solution, put the adsorption column CA2
  into the collection tube and repeat this step for once
* Place the adsorption column CA2 back to the collection tube, centrifuge (12000 rpm, 2 min)
* Dry the adsorption column CA2 at room temperature
* Place the adsorption column CA2 in a clean centrifuge tube, add elution buffer EB to the middle of the
  adsorption membrane, place it for 2min at room temperature
* Centrifuge (12000 rpm, 2 min)
* Collect DNA solution

#### 5. Homologous recombination ligation transformation

The PCR fragments and gel-recovered digestion vectors were ligated with the ClonExpress II One Step Cloning Kit,
a homologous recombinase from Novozan.

* Mix the digested vector, PCR fragments, and a certain amount of enzyme.
* The mixture reacts at 50°C for 30 min.
* Mix chemically competent cells with ligated plasmid DNA.
* Incubate the cells with DNA on ice for 30 min.
* Perform heat shock at 42°C for 45 s as appropriate for the bacterial strain and DNA used.
* Return the heat-shocked cells to the ice for more than 2 min.
* Prepare the medium with appropriate antibiotics.
* Prepare the cell suspension that is required to be cultured or screened.
* Spread cell suspension with a cell spreader.
* Incubate at 37℃ in an incubator for 12-16 h.
* The colonies are sent for sequencing.

#### 6. Protein expression purification

* Extract Amplified Bacterial Culture: After confirming the completion of amplification, add 500 ul of
  antibiotics (Amp) to 500ml culture LB liquid, remove the bacterial culture from the incubator, mix Amp with LB
  liquid together.
* Temperature Reduction Treatment: Place the bacterial culture in a 4°C refrigerator and set a timer（20 min） to
  ensure accurate cooling time. Simultaneously, adjust the shaker temperature to 16°C and begin preheating.
* Sterile Operation to Remove Seal Film: Within the laminar flow hood, ignite the alcohol burner and use sterile
  tweezers to swiftly and smoothly remove the seal film from the culture bottle beside the flame.
* Addition of IPTG to Medium: Precisely measure out 100μL of IPTG solution and carefully add it to the 500 ml
  liquid medium, thoroughly mixing to ensure uniformity.
* Inoculation of Bacterial Culture into Medium: Inoculate the cooled bacterial culture into the IPTG-containing
  medium at an appropriate ratio, gently mixing to combine.
* Low-temperature Cultivation: Transfer the inoculated medium to the preheated 16°C shaker, set the speed to
  180rpm, and conduct continuous cultivation for 18 hours.
* Establishment of Control Group:Prepare a group of BL21 strains without plasmids, perform a 10 ml shaking
  operation similarly, serving as the experimental control group.
* Cultivation of Bacterial Cells : Centrifuge the bacterial solution at 16℃ for 4000 g for 10 min, discard the
  supernatant and collect the bacteria until 1 l of bacterial solution is used up.
* Add 30 ml of purified buffer into the centrifuge tube to resuspend the cells.
* Put the suspended bacterial solution on the ice and set the ultrasonic crushing program 300 W to work for 10 s
  and stop for 10 s for 15 min.
* The solution was then centrifuged at 13000 g at 4℃ for 40 min, and the supernatant was collected.
* 2 ml nickel medium was added to the gravity column, 2 volumes of digestion buffer was added for balance, the
  supernatant passed through the column and the miscellaneous laundry operation will be performed.
* Plug the lower end of gravity column and add 3 ml digestion buffer.
* Add 5 μl Ppase and digest the enzyme for 12-16 h at 4℃ .
* Protein Elution and Collection: Wash the protein with 15 ml of purification buffer, collect eluted proteins
  into a tube, and store 40 μl of enzymatically cleaved nickel resin into two separate tubes for later use; and
  add 50 μl of G250 to 20 μl of bacterial culture (nsp5/nsp5\_native) to perform the dye test and measure protein
  concentration.
* Centrifugation: Centrifuge at 3600 g, 4°C for 10 min. Perform dye tests for each tube: nsp5 and nsp5\_native.
  Add 50 μl of coomassie brilliant blue G250 dye. Add 20 μl of supernatant into one test tube and 20μl of
  collected liquid in the column into another tube
* Store the remaining purified protein at -80°C

#### 7. SDS-PAGE

* Add melted 2×SDS (10 μL) loading into containers and heat around 100℃ in a water bath for 6 min
* Add SDS loading buffer to the buffer chamber
* Inject samples into the precast gel
  + Gel specifications: Tris-Glycine, 12%, 15 holes, 1.5 mm
* Conduct electrophoresis at 120 V for 60 min
  + Increased voltage above reccomended value to ensure maximal Marker performance
* Stain the gel with FETO SDS-PAGE staining buffer for 15 min, horizontal shaker 70 rpm
* Rinse with ddH2O twice

#### 8. Co-transformation and enzyme activity

Verify FlipGFP Cleavage and Fluorescence Relief Co-transformation allowed the simultaneous expression of both
split fluorescent protein fragments and nsp5 within the same host cell. This setup enabled nsp5 to cleave the
linker between the GFP fragments, leading to their correct assembly and the generation of a detectable
fluorescence signal. Without co-transformation, the fragments would remain unassembled, and fluorescence would not
occur. The presence of active nsp5 facilitated the proper assembly of GFP, confirming its crucial role in enabling
fluorescence.

#### 9. Inhibitors impact

By testing the co-transformation function, adding effective inhibitors and ineffective inhibitors to measure the
enzyme activity of the nsp5. Using ELISA to show the fluorescent signal to determine the substrate's
concentration. A decrease or loss of fluorescence indicated successful inhibition of the protease, preventing
cleavage of the linker and assembly of the fluorescent protein.

{% endblock %}

{% 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 %}

[Overview](#overview)
[Successful expression, purification and characterisation of SARS-Cov-2 nsp5 with native N-terminal and C-terminal.](#s1)
[Successful construction of FlipGFP that can be activated by nsp5.](#s2)
[Successful design of nsp5-T21I with enhanced enzyme activity.](#s3)

## Overview

Our team reached the following key results after four rounds of DBTL:

* Expression purification and enzyme activity characterisation of SARS-Cov-2 nsp5 with native N-terminal and C-terminal.* Construct a FlipGFP system that can be activated by nsp5.
  * Rational design of nsp5-T21I with enhanced enzyme activity.

## Successful expression, purification and characterisation of SARS-CoV-2 nsp5 with native N-terminal and C-terminal.

Through two rounds of DBTL for sequence design and optimisation, we successfully expressed and purified SARS-Cov-2 nsp5 in *E. coli* BL21.In order to accurately characterise nsp5 we determined the efficiency of nsp5 degradation of substrates using FRET and characterised the *kcat*/*Km* of nsp5 based on the Michaelis-Menten equation(Figure 1).

{{ figure(
image="https://static.igem.wiki/teams/5131/results/result-1.webp",
caption="Figure 1. Purification of nsp5\_native. Lane 1-3: marker, digested nickel beads, purified protein(left panel). Kinetic model of nsp5 enzyme activity(right panel).",
width="70%"
) }}

## Successful construction of FlipGFP that can be activated by nsp5

To ensure the correct expression of FlipGFP, we used the pRSF-Duet1 and separately inserted the first nine β-strands of FlipGFP and the engineered 10-11 β-strands into two different ORFs. We separately transformed *E. coli* BL21 with FlipGFP alone and co-transformed *E. coli* BL21 with FlipGFP and nsp5, then plated them on LB plates containing IPTG at a final concentration of 0.2 mM. The results showed that colonies transformed with FlipGFP alone produced almost no fluorescence, while colonies co-transformed with FlipGFP and nsp5 emitted significant fluorescence. This demonstrates that we successfully constructed a FlipGFP system that can be activated by nsp5 to emit fluorescence(Figure 2).

{{ figure(
image="https://static.igem.wiki/teams/5131/results/result-2.webp",
caption="Figure 2. Activation of FlipGFP by nsp5",
width="70%"
) }}

## Successful design of nsp5-T21I with enhanced enzyme activity.

Through rational design (see [Model](https://2024.igem.wiki/squirrel-shanghai/moddel) for details), we designed a mutant nsp5-T21I that may have higher enzyme activity than wild type. We first verified that this mutant could be solubilised for expression in *E. coli* BL21 and purified by nickel affinity chromatography(Figure 3).

{{ figure(
image="https://static.igem.wiki/teams/5131/results/result-3.webp",
caption="Figure 3. Purification of nsp5-T21I. Lane 1-2: marker,purified protein",
width="30%"
) }}

We determined the enzyme activity of nsp5-T21I by FRET. The results showed that nsp5-T21I had a higher *kcat*/*Km*(35,069 s⁻¹M⁻¹) compared to wild-type nsp5(27,691 s⁻¹M⁻¹), as well as a higher reaction rate at the same substrate concentration(Figure 4). This indicates that we successfully designed the nsp5 mutant with higher enzyme activity compared to the wild type.

{{ figure(
image="https://static.igem.wiki/teams/5131/results/result-4.webp",
caption="Figure 4. Kinetic model of nsp5-T21I(left panel). Reaction rates comparsion between WT nsp5 and nsp5-T21I(right panel).",
width="70%"
) }}

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