Molecular Biology and Genetic Tools to Investigate Functional Redundancy Among Fe-S Cluster Carriers in E. coli.
Methods Mol Biol
; 2353: 3-36, 2021.
Article
en En
| MEDLINE
| ID: mdl-34292541
Iron-sulfur (Fe-S) clusters are among the oldest protein cofactors, and Fe-S cluster-based chemistry has shaped the cellular metabolism of all living organisms. Over the last 30 years, thanks to molecular biology and genetic approaches, numerous actors for Fe-S cluster assembly and delivery to apotargets have been uncovered. In prokaryotes, Escherichia coli is the best-studied for its convenience of growth and its genetic amenability. During evolution, redundant ways to secure the supply of Fe-S clusters to the client proteins have emerged in E. coli. Disrupting gene expression is essential for gene function exploration, but redundancy can blur the interpretations as it can mask the role of important biogenesis components. This chapter describes molecular biology and genetic strategies that have permitted to reveal the E. coli Fe-S cluster conveying component network, composition, organization, and plasticity. In this chapter, we will describe the following genetic methods to investigate the importance of E. coli Fe-S cluster carriers: one-step inactivation of chromosomal genes in E. coli using polymerase chain reaction (PCR) products, P1 transduction, arabinose-inducible expression system, mevalonate (MVA) genetic by-pass, sensitivity tests to oxidative stress and iron starvation, ß-galactosidase assay, gentamicin survival test, and Hot Fusion cloning method.
Palabras clave
A-type carrier; Arabinose-inducible expression system; Electrotransformation; Fe-S cluster biogenesis; Gentamicin survival test; Hot Fusion cloning method; ISC; Iron-sulfur clusters; MVA genetic by-pass; One-step inactivation of chromosomal genes in E. coli using PCR products; P1 transduction; SUF; Sensitivity tests to oxidative stress and iron starvation; ß-Galactosidase assay
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Escherichia coli
Límite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2021
Tipo del documento:
Article
País de afiliación:
Francia
Pais de publicación:
Estados Unidos