The model for Nanobodies
Plasmid design
RFP – engineered mutant of red fluorescent protein from Discosoma striata (coral)
HlyA- C-terminal signal sequence of alpha-hemolysin
The construct was synthesized by GenScript company in pUC57 vector
We inserted the construct into the pSB1C3 plasmid into the standard restriction sites of EcoRI and PstI
Introducing permanent competence into E. coli
Making construct
The gene for Gp16 ATP-ase protein was ordered from GenScript company.Then, it was combined with the constitutive Anderson promoter + INP, and the constructed part was cloned into standard pSB1C3 plasmid with Circular polymerase extension cloning (CPEC).
References
Fraile, S., Muñoz, A., De Lorenzo, V., & Fernández, L. A. (2004). Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli. Molecular microbiology, 53(4), 1109-1121
Schwartz C, De Donatis GM, Fang H, Guo P. (2013). The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily. Virology. 443: 20–27.
Quan J, Tian J. (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. doi:10.1371/journal.pone.0006441
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