Plasmid

Part:BBa_K4861005

Designed by: Zhang Haobo   Group: iGEM23_BJHS   (2023-07-16)


pET-28a(+)-SARS-CoV-2-delta-RBD

The spike protein of the delta variant of the novel coronavirus is a key protein for infecting the human body. It enters host cells by binding to the ACE2 receptor on the surface of the host cells. Therefore, the S protein is preferred for designing recombinant protein vaccines against the novel coronavirus. The S protein is divided into two subunits, S1 and S2, with S1 containing a receptor-binding domain (RBD), which is a critical structure for the virus to enter host cells. We used the restriction enzymes NcoI and XhoI to digest the SARS-CoV-2 RBD gene and the vector. Subsequently, the SARS-CoV-2 RBD fragment was ligated to the pET-28a vector backbone using T4 DNA ligase. Finally, the recombinant plasmid was transformed into Escherichia coli for screening.


Delta-pET28a

Delta-pET28a

Construction Design

Delta-pET28a is a novel plasmid constructed using the pET-28a vector (BBa_K3521004) and a gene fragment Delta-SARS-CoV2 RBD (BBa_K4861001).

Figure 1
Figure 1: Delta-pET28a Plasmid

Engineering Principle

The S (spike) protein of neocoronaviruses is a key protein for their infection of the human body, which mediates viral entry into host cells by binding to the ACE2 receptor on the surface of the host cell. The S protein is divided into two subunits, S1 and S2, of which the S1 subunit contains a receptor-binding domain (RBD), which is a key structure when the virus enters the host cell. The code of Delta-SARS-CoV2 RBD represents a gene segment of the S protein from the Delta strain (COVID-19).

When this plasmid is introduced into BL21 Escherichia coli, it will produce the recombinant protein for manufacturing recombinant protein vaccines for viral strains.

Figure 2
Figure 2: Schematic Representation of the Engineering Principle

Experimental Approach

In the initial phase, we began with extracting the pET28a plasmid, which served as the foundational framework. Following this, we conducted double enzyme cuts on both the blank plasmid and the gene segments of viral strain –Delta– using NcoI and XhoI enzymes. After these cuts, we merged the target fragments from four viral strains with the pET28a plasmid using T4 DNA ligase. This intricate process transported the target fragments onto the blank plasmid.

Subsequently, we transferred the constructed plasmids into DH5α cells and subjected them to an overnight cultivation in a culture medium.

Figure 3
Figure 3: LB Medium of DH5α for Overnight Culture

Following that, we isolated the plasmids from the well-cultivated DH5α cells and subjected them to a second round of enzyme cuts, followed by gel electrophoresis to confirm the success of our plasmid construction. As depicted in figure 2, the gel electrophoresis results displayed two distinct bands at approximately 6000 base pairs and 760 base pairs. These bands corresponded respectively to the pET28a plasmid and the viral target segments.

Figure 4
Figure 4: Gel Electrophoresis Results

With the success of our plasmid construction confirmed in the previous phase, we moved forward to express and purify the proteins. As DH5α lacks the ability to express proteins, we transferred the constructed plasmids into BL21 bacteria for protein expression. Following an overnight cultivation (as shown in figure 3), we proceeded to the next steps.

Figure 5
Figure 5: LB Medium for BL21 Overnight Culture

Using the results from our modeling analysis, we decided to go with conditions involving 37°C and an IPTG concentration of 0.5mM for scaling up the culture. After expanding the cultivation, we once again went through the steps of ultrasonic disruption and centrifugation.

Consulting the scientific literature, we learned that the COVID-19 RBD protein is inclusion body protein. In simple terms, it's like a protein cluster that forms inactive solid particles within the cells. This can be seen in figure 6 with the red circle.

Figure 4
Figure 4: Inclusion Body Protein

Characterization/Measurement

Having obtained the purified protein, we proceeded to a crucial phase: the ELISA test. Our objective was to scrutinize the interaction between the RBD protein and the human ACE2 protein, a pivotal step in assessing the potential of our vaccine. This phase held the key to unveiling whether our project was on the right track.

Figure 5
Figure 5: Changing Color in Solution on the High-Binding Plate

Moving forward, we quantified our success by measuring the OD450 values. By subtracting the values of the control group (0mg/ml), we accentuated the true essence of the experiment. The graphical representation of this processed data not only shows the trend but also clarifies the impact of the interaction.

Figure 6
Figure 6: Results of ELISA

Reference

  1. N. Zhu et al., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382, 727-733 (2020).
  2. R. Lu et al., Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565-574 (2020).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4762
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4762
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4762
    Illegal BglII site found at 4402
    Illegal BglII site found at 4866
    Illegal XhoI site found at 5213
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4762
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4762
    Illegal NgoMIV site found at 2622
    Illegal NgoMIV site found at 2782
    Illegal NgoMIV site found at 4370
    Illegal AgeI site found at 4742
    Illegal AgeI site found at 5099
  • 1000
    COMPATIBLE WITH RFC[1000]


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