Difference between revisions of "Part:BBa K4389000"

Revision as of 10:19, 6 October 2022

B5R (all 4 sushi domains)

B5R

Biology

The B5R gene encodes 42-kDa glycosylated type I membrane protein of the envelope of the Vaccinia virus [1] (Figure 1). The protein B5R is highly conserved among multiple strains of vaccinia virus as well as in other orthopoxviruses, expanding the range of use for the detector of this protein [2]. We designed our cloning and expression strategy based on the three-dimensional (3D) structure of the B5 protein that we derived using AlphaFold2 software (Figure 2). Each of its four Sushi domains, which make up its ectodomain, has two intramolecular disulfide linkages [3]. The recombinant protein must be refolded, as we did for other Vaccinia viral proteins since inclusion bodies are unavoidably obtained.

Figure 1. Intracellular mature (IMV) and extracellular enveloped (EEV) orthopoxvirus [4]

Figure 2. Modeled 3D structure of B5R protein

Usage

According to published protocols, B5R protein was expressed in E. coli, hence we decided to follow a similar procedure [5]. PCR primer is designed and ordered for the amplification of the desired B5 sequence from the respective pVax1 constructs (Figure 3), and specific restriction sites that are compatible with the pET23a vector (Figure 4) (NdeI at the 5’ end and XhoI at the 3’ end) will be added. The readily available pET-23a plasmid will be utilized for E.coli overexpression since it possesses a C-terminal 6His-tag, optimal for this cloning and further purification.

Figure 3. pVAX plasmid structure

Figure 4. pET plasmid structure

Part Funcionalization

Visualization

Gel electrophoresis (PCR products of 1st sushi and 4 sushi encoding DNA)

Figure 1 shows the picture of agarose gel run after PCR amplification and subsequent PCR purification of genes obtained from pVax1 B5R plasmid which are later to be inserted into an expression construct, pET23a plasmid. Gel electrophoresis provided confirmation of the successful amplification of genes encoding 1st sushi domain of protein B5R (A) and all 4 sushi domains of B5R protein (B).

Figure 1. Results for gel electrophoresis of PCR products

E. coli DH5α cells were transformed with the ligation products of double-digested pET23a plasmid and sequences encoding 1st sushi domain (Figure 3A) and 4 sushi domains (Figure 3B). For the positive control, cells were transformed with the pUC control plasmid. For the negative control, the cells were not transformed prior to plating. The cells were grown in a selective medium of Luria Bertani agar and Ampicillin (100 mg/mL). Plasmid extraction was performed to amplify the number of ligation products, which are later to be used to transform E. coli BL-21 cells for the expression of protein B5R.

Figure 3. Transformation of E. coli DH5α cells with ligation products: A) E. coli DH5α cells transformed with ligation product of pET23a plasmid and sequence encoding 1st sushi domain B) E. coli DH5α cells transformed with ligation product pET23a plasmid and sequence encoding 4 sushi domains C) Positive control: E. coli DH5α cells transformed with pUC control plasmid only D) Negative control: Non-transformed E. coli DH5α cells

The sequencing results were checked for alignment with the original plasmid DNA for the presence of 1st sushi and all 4 sushi domains’ parts in the colony's DNA for further plasmid extraction. As can be seen from Figure 7 all samples have alignment with the original DNA sequences encoding 1st sushi and all 4 sushi domains. The sequencing chromatogram confirms the proper cloning of 1st sushi and all 4 sushi domains into the E.coli pET23a plasmid.

Figure 8. Sequence alignment with plasmid DNA for 1st sushi and all 4 sushi domains. Image created in SnapGene.

Sequence and Features