Plasmid

Part:BBa_K4862004

Designed by: Zhang Boxuan   Group: iGEM23_BZK-SH   (2023-08-21)


pET28a-NS3 hel


Composite Part: BBa_K4862004 (pET-NS3-Hel)

Composite Part: BBa_K4862004 (pET-NS3-Hel)

Construction Design

We designed enzyme digestion to connect the Hel gene with the PET28a (BBa_K3521004) plasmid. To do that, we first obtained the NS3-Hel (BBa_K4862000) through PCR cloning. Then, we used double enzyme digestion with BamHI, XhoI, CutSmart, and ddH2O to connect the Hel gene with PET28a.

Construction design of plasmid pET-NS3-Hel
Figure 1. Construction design of plasmid pET-NS3-Hel

Engineering Principle

The helicase domain of NS3 (NS3 hel), together with NS5, an RNA-dependent RNA polymerase, participates in viral RNA replication and is essential for genome propagation. It has been demonstrated that the interaction between DENV NS3 hel and NS4B triggers the dissociation of the helicase from single-stranded RNA, thereby modulating viral replication[1]. NS3 protein is one of the most highly conserved proteins in flaviviruses, with multiple enzymatic activities [5].

RNA helicase is responsible for almost all metabolism of RNA; in the Dengue virus, NS3 protein contains the activity of multiple enzymes, including helicase. Inserting the NS3 sequence is for expressing the protein we need and testing whether the medicine finally injected would be effective. The other structure of the pET28a plasmid would not be changed.

Experimental Approach

Per the construction design, we obtained the plasmid pET-NS3-Hel. To express the DNA as a protein, we used heat shock conversion to insert PET28a-NS3-Hel into BL21(DE3), a type of Escherichia coli. After this, we transplanted the bacteria by touching the single colony linings of PET-NS3-Hel with the pipette gun and leaving the pipette in the LB broth for the 16-hour incubation.

Then, we amplified our target Hel DNA sequence by PCR and extracted the plasmid with buffer SP1, SP2, SP3, washing solution, and water. Electrophoresis gel was conducted to validate the successful result; as shown, the strip sizes of PET-NS3-Hel were detected in the 1344bp round. In addition, the sequencing result also supported this conclusion.

Electrophoresis gel and sequencing of pET-NS3-Hel
Figure 2. Electrophoresis gel and sequencing of pET-NS3-Hel

Since we have obtained the BL21 pET-NS3-Hel, firstly we need to check if this part pET-NS3-Hel could work as expected to express the protein. We used IPTG to express the Hel gene through BL21(DE3) while preparing the 15% SDS-PAGE gel. We broke the bacteria with an ultrasonic cell breaker device. We collected the unpurified Hel protein solution by adding HIS Buffer A to the centrifuge. To purify the protein solution, we utilized Ni-NTA His Tag Purification. Per the SDS-PAGE shown in Figure 4, the protein NS3-Hel was detected at around 49 kDa.

SDS-PAGE gel indicating NS3-Hel’s kDa value
Figure 3. SDS-PAGE gel indicating NS3-Hel’s kDa value

Characterization/Measurement

Secondly, we further used the FRET method to check if the protein NS3-Hel possesses helicase activity. We added fluorescence and ATP into the NS3-Hel pure protein solution to test whether the helicase is inactive. According to the pET-28a-NS3-Hel curve compared to the control pET-28a, the fluorescence value decreases, whereas protein concentration increases. Therefore, we can prove that the NS3 protein possesses helicase activity.

FRET curves of pET-28a-NS3-Hel and pET-28a
Figure 4. FRET curves of pET-28a-NS3-Hel and pET-28a

*Our team measured the fluorescence using fluorometers with an excitation of 620 nm and an emission wavelength of 685 nm.

References

  1. de Borba L, Strottmann DM, de Noronha L, Mason PW, Dos Santos CN. Synergistic interactions between the NS3(hel) and E proteins contribute to the virulence of dengue virus type 1. PLoS Negl Trop Dis. 2012;6(4):e1624. doi: 10.1371/journal.pntd.0001624. Epub 2012 Apr 17. PMID: 22530074; PMCID: PMC3328427.
  2. Kapoor M, Zhang L, Ramachandra M, Kusukawa J, Enber KE, et al. (1995). The association between NS3 and NS5 proteins of DENV2 in the putative RNA replicase is linked to differential phosphorylation of NS5. J Biol Chem 270:19100–19106.
  3. Westaway EG, Mackenzie JM, Khromykh AA (2003) Kunjin RNA replication and applications of Kunjin replicons. Adv Virus Res 59: 99–140.
  4. Umareddy I, Chao A, Sampath A, Gu F, Vasudevan SG (2006) Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA. J Gen Virol 87: 2605–2614.
  5. Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, et al. (2009). Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol 83: 12895–12906.
  6. Bollati M, Alvarez K, Assenberg R, Baronti C, Canard B, et al. (2010) Structure and functionality in flavivirus NS-proteins: Perspectives for drug design. Antiviral Res 87: 125–148.


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 5746
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3966
    Illegal NgoMIV site found at 4126
    Illegal NgoMIV site found at 5714
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 455
    Illegal BsaI.rc site found at 835
    Illegal BsaI.rc site found at 1109
    Illegal SapI.rc site found at 802


[edit]
Categories
Parameters
None