Team:Queens Canada/Results

Cloning

The biobrick was ordered from IDT, digested, and ligated into pET24d (Fig. 1). pET24d was chosen as a vector due to the availability of the T7 promoter.

Figure 1. Test digest of ScFv-mNG integration into the pET24d vector. The band above 1.5 kb indicates successful integration of the 1536 bp biobrick.

Expression

The biobrick was expressed in E. coli Rosetta gami2, as this cell line contains an oxidizing cytoplasmic environment ideal for the disulfide bond formations within the antibody (Fig. 2). Following a nickle affinity column purification, the ScFv was observed at 56 kDa. Note that another biobrick (BBa_K3056001) was designed, with EGFP as the fluorescent protein, rather than mNG, to compare fluorescent intensities.

Figure 2. Nickel column purification of ScFv-mNG and ScFv-EGFP. Both proteins were successfully purified from BL21 cells, as indicated by the major band at 56 kDa.

THC Binding

The purified EGFP and mNG linked ScFv were spot tested on a membrane assay, where only the ScFv was able to bind the THC soaked membrane (Fig. 3). To test the binding, lipophilic membranes were saturated with 10 mg/mL of THC, washed 10x with phosphate buffered saline (1% Tween-20), incubated with ScFv-EGFP, or ScFv-mNG, washed 3x with phosphate buffered saline (1% Tween-20), and imaged in Azure Biosystems 600. The excitation and emission wavelengths were 395 nm, and 509 nm, respectively.

Figure 3. THC membrane assay using purified ScFv-EGFP and ScFv-mNG. Membranes were saturated with 10 mg/mL of THC, washed, and probed with the purified ScFv. Only ScFv-mNG gave a positive signal in the assay