Plasmid

Part:BBa_K4861007

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


pET-28a(+)-SARS-CoV-2-XBB1.5-RBD

The spike protein of the XBB1.5 variant of the COVID-19 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 the preferred choice 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.


XBB1.5-pET28a BBa_K4861007

XBB1.5-pET28a BBa_K4861007

Construction Design

We ligated a segment of XBB1.5-SARS-CoV2 RBD gene sequence (BBa_K4861003 - parts.igem.org) to the original part (BBa_K3521004 - parts.igem.org) to obtain the new part (BBa_K4861007). In this project, our ultimate goal is to produce a recombinant protein vaccine against COVID-19. Therefore, we plan to produce related proteins encoded by the XBB1.5-RBD sequence through engineering bacteria.

XBB1.5-pET28a BBa_K4861007

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, and the S protein is preferred for the design of recombinant protein vaccines against neocoronaviruses[1]. The S protein is divided into two subunits, S1 and S2, of which the S1 subunit contains a receptor-binding domain (RBD, receptor- binding domain), which is a key structure when the virus enters the host cell. Therefore, it is very appropriate to choose RBD as the target antigen for designing recombinant protein vaccines[2]. The code of XBB1.5-SARS-CoV2 RBD represents a gene segment of the S protein from the XBB1.5 strain (COVID-19).

When this plasmid is introduced into BL21 Escherichia coli, it will produce the recombinant protein we need. It can be used to manufacture recombinant protein vaccines for viral strains.

XBB1.5-pET28a BBa_K4861007

Cultivation, Purification and SDS-PAGE

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 –XBB.1.5 – 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 overnight cultivation in a culture medium. The objective behind this step was to foster a vast yield of the constructed plasmids through DH5α cloning.

LB medium of DH5α for overnight culture

Figure 1. 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.

Enzyme digestion validation of recombinant plasmids

Figure 2. Enzyme digestion validation of recombinant plasmids

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.

LB medium for BL21 overnight culture

Figure 3. 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 inclusion body protein. In simple terms, it's like a protein cluster that forms inactive solid particles within the cells.

Inclusion body protein

Figure 4. Inclusion body protein

Function Testing

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. In a meticulous sequence, we finished the ELISA test within a high-binding plate. Sequentially, we introduced the components – RBD protein, Biotinylated-ACE2, Streptavidin-HRP, and the TMB substrate solution. As this chemical symphony unfolded, a discernible change in solution color emerged, signifying the culmination of our experiment and its intrinsic success.

Changing color in solution on the high-binging plate

Figure 5. Changing color in solution on the high-binging 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.

Results of ELISA

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 characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565-574 (2020).


Sequence and Features


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


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