Difference between revisions of "Part:BBa K343000"

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=== Background ===  
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=FlhDC master operon=
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== Part 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)]
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More than 80% of all known bacterial species express flagella [https://parts.igem.org/Part:BBa_K343004#References [1]]. The synthesis of the flagella is a complex and energy consuming process, so the expression of the involved proteins is tightly regulated by the extracellular environment. One of the most well studied flagella synthesis systems is that of ''Eschericia coli''. Here the expression of the master regulator ''FlhDC'' operon is controlled i.e. by pH, salt concentration or the availability of nutrients. Apart from initiation of flagella synthesis FlhDC is also a repressor of cell metabolism as cell growth and flagellation does not occur at the same time. FlhDC is a hexameric transcription factor that consists of four FlhD subunits and two FlhC subunits (FlhD<sub>4</sub>C<sub>2</sub>). It constitutes the first class in the three classed flagella synthesis cascade. Class II consist of genes encoding proteins that make up the basal body, as well as the alternative transcription factor, σ<sup>28</sup>, which is responsible for the expression of the class III genes. These genes in turn encodes the proteins that composes the tail filaments.
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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)]
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Studies of insertion elements (IS) upstream of the ''flhDC'' operon, has shown that an upregulation of the expression of the operon is responsible for the motility of the ''E. coli'' MG1655 strain. [https://parts.igem.org/Part:BBa_K343004#References [2]]It has also been shown that overexpression of the ''FlhDC'' operon restores motility in mutants that have been made immotile [https://parts.igem.org/Part:BBa_K343004#References [3]]. <br><br>
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The native FlhDC coding sequence (K343100) has an internal Pst1 site located 822bp into the operon. This has been replaced by a silent T to C mutation.
 
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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)]
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=Refrences=
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# O. Soutourina , PN. Bertin [http://www.ncbi.nlm.nih.gov/pubmed/14550943 Regulation cascade of flagellar expression in Gram-negative bacteria.]<br>
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# O. Soutourina, A. Kolb, E. Krin, C. Laurent-Winter, S. Rimsky, A. Danchin, and P. Bertin[http://jb.asm.org/cgi/content/short/181/24/7500 Multiple Control of Flagellum Biosynthesis in Escherichia coli: Role of H-NS Protein and the Cyclic AMP-Catabolite Activator Protein Complex in Transcription of the flhDC Master Operon]<br>
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# Eric J. Gauger, Mary P. Leatham, Regino Mercado-Lubo, David C. Laux, Tyrrell Conway, and Paul S. Cohen [http://iai.asm.org/cgi/content/abstract/75/7/3315 Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine{triangledown}]<br>
 
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===Usage and Biology===
 
===Usage and Biology===
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This is only a coding sequence,
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 22:45, 27 October 2010

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.

FlhDC master operon

Part background


More than 80% of all known bacterial species express flagella [1]. The synthesis of the flagella is a complex and energy consuming process, so the expression of the involved proteins is tightly regulated by the extracellular environment. One of the most well studied flagella synthesis systems is that of Eschericia coli. Here the expression of the master regulator FlhDC operon is controlled i.e. by pH, salt concentration or the availability of nutrients. Apart from initiation of flagella synthesis FlhDC is also a repressor of cell metabolism as cell growth and flagellation does not occur at the same time. FlhDC is a hexameric transcription factor that consists of four FlhD subunits and two FlhC subunits (FlhD4C2). It constitutes the first class in the three classed flagella synthesis cascade. Class II consist of genes encoding proteins that make up the basal body, as well as the alternative transcription factor, σ28, which is responsible for the expression of the class III genes. These genes in turn encodes the proteins that composes the tail filaments.

Studies of insertion elements (IS) upstream of the flhDC operon, has shown that an upregulation of the expression of the operon is responsible for the motility of the E. coli MG1655 strain. [2]It has also been shown that overexpression of the FlhDC operon restores motility in mutants that have been made immotile [3].

The native FlhDC coding sequence (K343100) has an internal Pst1 site located 822bp into the operon. This has been replaced by a silent T to C mutation.

Refrences


  1. O. Soutourina , PN. Bertin [http://www.ncbi.nlm.nih.gov/pubmed/14550943 Regulation cascade of flagellar expression in Gram-negative bacteria.]
  2. O. Soutourina, A. Kolb, E. Krin, C. Laurent-Winter, S. Rimsky, A. Danchin, and P. Bertin[http://jb.asm.org/cgi/content/short/181/24/7500 Multiple Control of Flagellum Biosynthesis in Escherichia coli: Role of H-NS Protein and the Cyclic AMP-Catabolite Activator Protein Complex in Transcription of the flhDC Master Operon]
  3. Eric J. Gauger, Mary P. Leatham, Regino Mercado-Lubo, David C. Laux, Tyrrell Conway, and Paul S. Cohen [http://iai.asm.org/cgi/content/abstract/75/7/3315 Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine{triangledown}]


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]