Part:BBa_K3767002

Components: This part consists of a developed ScFv sequence (BBa_K3767000) based on previous research conducted by Ghosh and Huber ^[1] using SAbPred’s ABodyBuilder, Clusterpro supercomputer and PyMol to determine if the ScFv would bond to the target OspA surface protein. The part also consists of an improved Alkaline Phosphatase(BBa_K3767000) linked to the ScFv sequence via (GGGS)3 linkers at the C-terminal.

Background: Alkaline Phosphatases (phoA) are ubiquitous membrane bound glycoprotiens that catalyze the hydrolysis of phosphate monoesters at basic pH levels with release of inorganic phosphate^[2]. They are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization^[3]. The phoA was linked to the ScFv sequence as a chromogenic reagent, meaning the when the phoA is mixed with with 5-Bromo-4-Chloro-3-indolyl phosphate (BCIP) expresses a blue color in the 615 nm range which is visible to the naked eye. Alkaline phosphatase is linked to the C-terminus of the ScFv’s light chain through (GGGS) [glycine] linkers. Thus, the ScFv is responsible for binding to OspA and will only express color if once the ScFv binds to the target OspA protein.

References

1. Ghosh, S., and Huber, B. T. (2007) Clonal diversification in OspA-specific antibodies from peripheral circulation of a chronic Lyme arthritis patient. J. Immunol. Methods. 321, 121–134

2. Sharma,U., Pal,D., and Prasad,R. (2013) Alkaline Phospatase: An Overview, Indian J Clin Biochem, [Online] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062654/, (accessed June 12 2021)

3. Part:BBa K1216001 - parts.igem.org [online] https://parts.igem.org/Part:BBa_K1216001#References (Accessed June 12, 2021) 

4. Du, M. H. L., Lamoure, C., Muller, B. H., Bulgakov, O. V., Lajeunesse, E., Ménez, A., and Boulain, J. C. (2002) Artificial evolution of an enzyme active site: Structural studies of three highly active mutants of Escherichia coli alkaline phosphatase. J. Mol. Biol. 316, 941–953