Modification of the genome of Rhodobacter sphaeroides and construction of synthetic operons

Methods Enzymol. 2011;497:519-38. doi: 10.1016/B978-0-12-385075-1.00023-8.

Abstract

The α-proteobacterium Rhodobacter sphaeroides is an exemplary model organism for the creation and study of novel protein expression systems, especially membrane protein complexes that harvest light energy to yield electrical energy. Advantages of this organism include a sequenced genome, tools for genetic engineering, a well-characterized metabolism, and a large membrane surface area when grown under hypoxic or anoxic conditions. This chapter provides a framework for the utilization of R. sphaeroides as a model organism for membrane protein expression, highlighting key advantages and shortcomings. Procedures covered in this chapter include the creation of chromosomal gene deletions, disruptions, and replacements, as well as the construction of a synthetic operon using a model promoter to induce expression of modified photosynthetic reaction center proteins for structural and functional analysis.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Gene Deletion
  • Gene Expression Regulation, Bacterial
  • Genome, Bacterial*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Multigene Family
  • Operon*
  • Photosynthesis / physiology
  • Photosynthetic Reaction Center Complex Proteins / genetics
  • Photosynthetic Reaction Center Complex Proteins / metabolism
  • Rhodobacter sphaeroides / genetics*
  • Rhodobacter sphaeroides / metabolism
  • Synthetic Biology / methods

Substances

  • Bacterial Proteins
  • Membrane Proteins
  • Photosynthetic Reaction Center Complex Proteins