Plasmid

Part:BBa_K4843002

Designed by: Wu Delu   Group: iGEM23_TSB-Center   (2023-08-07)


pET28a-I7L

Composite Part: BBa_K4843002 (pET28a-i7L)

Composite Part: BBa_K4843002 (pET28a-i7L)

Construction Design

We designed two genes, I7L (BBa_K4843000), and extracted the plasmid pET28a (BBa_K3521004) from Escherichia coli. We then amplified the I7L, and the target DNA was obtained by PCR. The I7L genes are inserted into pET28a to get a recombinant plasmid. Then, the I7L-pET28a plasmid was transformed into BL21 (DE3) and DH5α by heat shock method.

DNA map of pET-28a-I7L
Figure 1. DNA map of pET-28a-I7L

Engineering Principle

According to the literature, within the replication cycle of the model vaccinia virus, a fundamental step is the maturation of non-infective virions from immature intracellular virions (IV) to mature intracellular virions (IMV). This maturation process is linked to the activity of the viral protease I7L, which is responsible for the cleavage of A17, a structural protein involved in the formation of the crescent membrane that eventually leads to the virion assembly. This cysteine protease is also responsible for the cleavage of several core proteins, including P25K/VP8, P4a, and P4b, which form more than 30% of the mass of the virion [1-2].

Experimental Approach

As shown in Figure 3, I7L obtained the correct genes we needed by confirming the length of bp which is 1269bp, respectively. So we can make this a prerequisite for further action.

Electrophoresis gel results after PCR
Figure 2. Electrophoresis gel results after PCR
Electrophoresis gel validation results
Figure 3. Electrophoresis gel validation results: 1: I7L; 2: I7L digested fragments; 3: ASFV-I7L; 4: ASFV-I7L digested fragments; 5: pET28a; 6: pET28a digested fragments

Plasmid Sequencing

To ensure that I7L fragments have been inserted into the plasmid in the right direction and there are no mutations during the heat shock process, we sent the successfully constructed plasmids for PCR identification and sequencing. Sequencing using template primers showed that the I7L fragments were inserted into the plasmid in the correct orientation. No mutations were found in the sequencing files (Figure 4), and the sequences were consistent with the sequences of Monkeypox virus (I7L) by NCBI blast comparison2.

Sequencing results
Figure 4. Sequencing results

Protein Expression and Purification

We validated protein purification production from the recombinant plasmid by Western Blot. As shown in Figure 5, the proteins I7L with the correct molecular weight (47kD) were obtained by purification of the two proteins with His-Tag. The protein markers used were Beyotime 14.4-116 protein markers.

Western Blot results
Figure 5. Western Blot results

Characterization/Measurement

In order to evaluate the activity of our purified proteins, we applied the FRET assay for function validation. After the purified protein was configured, 0.4 mM EDTA, 4 mM DTT, and 20% glycerol were added into the reaction buffer containing 20 mM HEPES, 120 mM NaCl, and pH 6.5. Then, 100 nM I7L was added into two parts, respectively. Finally, the blank control group was set up with BSA protein.

table 1

The linear curves in Figure 6 show that the fluorescence values of the two groups of target proteins decreased with the increase of time, and the control protein (BSA protein) remained unchanged with the increase of time, demonstrating that I7L was biologically active3.

Fluorescence assay results
Figure 6. Our team measured fluorescence using an enzyme labeler with an excitation wavelength of 320 nm and an emission wavelength of 380 nm3.

In summary, we successfully designed and validated I7L DNA parts, and inserted these genes into plasmid pET28a from Escherichia coli by enzyme digestion, enzyme-linked plasmid construction, and thermal excitation. We then transferred recombinant plasmids I7L into BL21, and the protein expression of the recombinant plasmid was induced by 0.4 mM IPTG, and the proteins were purified by His-Tag to obtain two pure proteins. Finally, the I7L biological activity was confirmed by fluorescence assay.

References

  1. Liu, L.; Cooper, T.; Howley, P.M.; Hayball, J.D. From Crescent to Mature Virion: Vaccinia Virus Assembly and Maturation. Viruses. 2014, 6, 3787.
  2. Byrd, C.M.; Hruby, D.E. Vaccinia Virus Proteolysis—A Review. Rev. Med. Virol. 2006, 16, 187–202.


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 BglII site found at 5549
    Illegal BamHI site found at 4971
  • 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
  • 1000
    COMPATIBLE WITH RFC[1000]


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