Difference between revisions of "Part:BBa K4477014"

Latest revision as of 02:42, 14 October 2022

Anti-mouse camelid VHH - complete expression cassette

Overview

This sequence codes for a heavy chain variable fragment (VHH), also known as a nanobody, that binds to the Fc region of mouse (murine) IgGs. Team Virginia 2022 designed this VHH for use as a secondary antibody to capture the hybrid human/mouse anti-oxLDL antibody we designed (BBa_K4477011). This VHH will serve excellently as a secondary antibody in a wide array of laboratory procedures such as Western blot for teams whose primary antibodies are murine in origin, among other potential applications.

The sequence of the VHH contains a promoter, RBS, coding sequence of the nanobody, and terminator; a complete expression cassette. Like the other composite parts designed by Virginia 2022, this part is designed for expression by IPTG induction. First, a strong T7 promoter occurs on the 5' end of the DNA sequence, facilitating IPTG induction. Then, an RBS the team optimized using the Salis RBS calculator for expression of the nanobody coding sequence occurs. The coding sequence for the nanobody occurs eight base pairs downstream of the 3' end of the RBS for optimal ribosomal translation. Lastly, a two-way terminator completes the composite part; as a two-way terminator, it not only prevents RNA polymerases from reading past the composite part into the backbone, but prevents any genes in the backbone from being transcribed into the composite part and interfering with their expression. Note that between each subcomponent, legal restriction enzyme sites were included for modularity.

Figure 1. Plasmid map.

Usage and Biology

This VHH antibody fragment is an anti-mouse IgG1 Fc-specific nanobody, a single-domain antibody derived from camelid heavy-chain antibodies (VHH). It is a secondary antibody that binds to the kappa chain, is monovalent, and does not cross-link with primary antibodies. This VHH has better detection signals than a commonly used commercially available polyclonal secondary antibody (1).

The VHH is intended to serve as a HRP-conjugated secondary antibody to be produced in E. coli. This allows current polyclonal secondary antibodies produced from animal immunization to be replaced with antibodies produced in E. coli, a much more sustainable and standardized method for the industry.

Characterization

Biobricking

We successfully BioBricked our VHH-encoding device.

Figure 2. Restriction Digest Verification of VHH Plasmid Construct. (A) Virtual digest with PstI and XbaI on Benchling yields two bands of sizes 2.6 kb and 0.6 kb. (B) 2: VHH colony cut with PstI and XbaI, 2.6 kb and 0.6 kb. 7: pSB3K3+mRFP cut with PstI, 3.7 kb. 8: pSB3K3+mRFP cut with XbaI, 3.5 kb. (C) VHH colony uncut, approx. 1.8 kb. 7: pSB3K3+mRFP uncut, approx. 2 kb.

After transformation of pSB3K3+VHH ligation product into DH5-alpha, plasmids were extracted from 6 white colonies and 1 pink colony for digestion analysis. The white VHH colony highlighted in Figure 2 (B) and (C) exhibited the same bands as in the virtual digest when cut with the same enzymes. The uncut colony shows a band lower on the gel than the linearized plasmid, which is expected for an uncut plasmid and indicates the plasmid preparation occurred successfully. The enzyme cutter controls we ran on pSB3K3+mRFP in lanes 7(B), 8(B), and 7(C) linearized pSB3K3+mRFP as expected, indicating the enzymes were functioning properly.

The successful results of the verification gel were further supported by sequencing data. The extracted VHH plasmid was found to be 97.47% similar to the theoretical VHH plasmid, indicating successful creation of our VHH-encoding device.

Sequence and Features