All-Codon Mutagenesis for Structure-Function Studies of Chemotaxis Signaling Proteins

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)

Abstract

The technique of all-codon mutagenesis can generate mutants that represent all possible amino acid replacements at any particular residue in a protein. It is thus a powerful tool to probe structure-function relationships in proteins of interest. In this chapter, we describe how we used all-codon mutagenesis to obtain mutants of the Escherichia coli serine receptor Tsr with amino acid replacements at residue F373, a functionally important site in this protein. We provide general protocols for mutagenesis of a target codon in a plasmid-borne gene and for the selection and screening of the resultant mutants. These techniques should be adaptable for the study of a variety of bacterial proteins.

Keywords

Chemoreceptor Chemotaxis Counter-selection Polymerase chain reaction Site-directed mutagenesis 

Notes

Acknowledgment

Our work is supported by research grant GM19559 from the National Institute of General Medical Sciences. DNA sequencing and primer synthesis were carried out by the Protein-DNA Core Facility at the University of Utah, which receives support from National Cancer Institute grant CA42014 to the Huntsman Cancer Institute.

References

  1. 1.
    Kim KK, Yokota H, Kim SH (1999) Four-helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor. Nature 400:787–792CrossRefGoogle Scholar
  2. 2.
    Ames P, Studdert CA, Reiser RH, Parkinson JS (2002) Collaborative signaling by mixed chemoreceptor teams in Escherichia coli. Proc Natl Acad Sci U S A 99:7060–7065CrossRefGoogle Scholar
  3. 3.
    Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid. J Bacteriol 134:1141–1156PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of BiologyUniversity of UtahSalt Lake CityUSA

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