Generation of Antibody Libraries for Phage Display: Preparation of Electrocompetent E. coli .

Autor:	Peng H; Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, Florida 33458, USA., Rader C; Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, Florida 33458, USA rader33458@gmail.com.
Jazyk:	angličtina
Zdroj:	Cold Spring Harbor protocols [Cold Spring Harb Protoc] 2024 Oct 15. Date of Electronic Publication: 2024 Oct 15.
DOI:	10.1101/pdb.prot108599
Abstrakt:	The size of an antibody library, that is, the phage display-selectable diversity, is restricted mainly by its transformation into the host bacterial cells. Electroporation is the most efficient method for transforming Escherichia coli with plasmids, including phagemids. Here, we describe the preparation of electrocompetent E. coli for the generation of phagemid-encoded antibody libraries encompassing 10 9 -10 11 independent transformants. To become electrocompetent, the bacterial suspension has to have high resistance, i.e., low ionic strength, which is achieved by gradually and gently transferring bacteria grown to mid-log phase to 10% (v/v) glycerol in highly pure water. The electrocompetent E. coli must be F plasmid-harboring bacteria, referred to as F + or male, in order to express F pili and be susceptible to infection by filamentous phage during library generation. In addition, it is necessary to apply antibiotic (e.g., tetracycline) pressure to retain the F plasmid, as it tends to segregate from bacteria. This protocol also includes assays for analyzing the prepared electrocompetent E. coli for competency, and evaluating potential contamination with helper phage, phagemid and phagemid-derived filamentous phage, and lytic phage. (© 2024 Cold Spring Harbor Laboratory Press.)
Databáze:	MEDLINE
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