Team:Queens Canada/Results
Results
Cloning
Our DNA was designed using Benchling before being order from IDT. Once received, the DNA was PCR amplified and digested using Xho1 and BamH1 before being ligated into a pET-28 a (+) vector. We opted to use the pET-28 a (+) vector due to the presence of a T7 promoter as well as the included His tag that would be attached following expression.
MP1 = Miniprep1
MP2 = Miniprep 2
EP = Empty Plasmid
U = Undigested
K = Digested with Kpn1
K + X = Digested with Kpn1 and Xho1
Expression
The ScFv antibody was expressed in E. Coli Rosetta-gami 2, as this cell line contains an oxidizing cytoplasmic environment which allowed for the disulfide bond formations within the antibody. In contrast, the OspA ligand was expressed in E. Coli BL21 cells lines as described by Chang et al. (1). Following nickel affinity column purification, we confirmed the successful expression of the 3-24 antibody as shown in figure two.
L = Ladder
E = Elution
P = Pellet
S = Supernate
TCL = Total Cell Lysate
Binding Testing
Final plans consisted of conducting ELISA binding testing of the OspA ligand to the 3-24 ScFv antibody by anchoring the ligand to a 96-well plate and washing the antibody over it and testing fluorescence of the GFP. Unfortunately, due to complications during cloning and expression which required extensive troubleshooting, we were unable to successfully testing practical antibody-ligand binding during our time in the lab.
References
1. Chang, Y.-F., Lauderdale, T.-L., Lee, W. Y., Shin, S. J., Jacobson, R. H., Appel, M. J., and Lein, D. H. (1993) Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli. FEMS Microbiol. Lett. 109, 297–301