Part:BBa_K343000

Mutated flagella master regulator (FlhDC mut).

The master regulator operon for flagellum synthesis. Produces transcriptional factors for Class II flagellar regulon genes. To make the part conform to the iGEM assembly standards we introduced a silent mutation, in an unintended restriction site, at BP 822 from t to c, so that Pst1 would not cleave the DNA there.

Background

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The flagella regulon in E. coli is composed of at least 50 genes organized in no less than 14 operons that all contribute to the synthesis and operation of flagella. The operons are synthesized in a three-level transcriptional cascade where the FlhDC operon is the master regulator at the top of the cascade. The flagella regulon is tightly controlled by nutritional and environmental conditions, E. coli starved of amino acids showed temporarily decrease of the flagella regulon transcripts which are needed for the synthesis and operation of the flagellum.[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06939.x/full (1)] The synthesis and assembly of flagella are regulated by the transcriptional cascade composed of three levels of gene products (class I, -II and –III). Class I genes consist of a single operon encoding the proteins FlhD and FlhC that form a multimeric (FlhD4C2) transcriptional activation complex. This ‘master regulator’ stimulates transcription by binding upstream of Class II promoters. Class II genes encode proteins that assemble to form the basal body and hook of the flagellum, as well as the fliA gene that encodes the alternative σ factor σ28, also called σF. σ28 binds to RNA polymerase (RNAP) core enzyme and directs it to Class III promoters. Class III genes encode the rest of the structural genes of the flagellum, including fliC encoding flagellin, as well as the chemotaxis apparatus. [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06939.x/full (1)]
It has been shown that overexpression of the FlhDC operon restores motility in mutants that have been made immotile [http://jb.asm.org/cgi/content/short/181/24/7500 (2)]. Also, overexpression of FlhDC in the E. coli K12 strain MG1655 made the cells hypermotile.[http://iai.asm.org/cgi/content/abstract/75/7/3315 (3)]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 609
  
• 1000
  COMPATIBLE WITH RFC[1000]

Safety

[http://iai.asm.org/cgi/reprint/75/7/3315 Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine]

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC93725/ The Yersinia enterocolitica Motility Master Regulatory Operon, flhDC, Is Required for Flagellin Production, Swimming Motility, and Swarming Motility]

[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0008029 Repression of Invasion Genes and Decreased Invasion in a High-Level Fluoroquinolone-Resistant Salmonella Typhimurium Mutant]