Difference between revisions of "Part:BBa K535004:Design"

(→‎Design Notes)
 
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**AACTGCAGCGGCCGCTACTAGTATCTTTCGTTCCATCCATCGCTGAG
 
**AACTGCAGCGGCCGCTACTAGTATCTTTCGTTCCATCCATCGCTGAG
  
−
† and has no modifications according to the reported sequence by Emmanuel Salazar, 2010.
 
  
 
===Source===
 
===Source===

Latest revision as of 23:36, 27 September 2011

NifH promoter -> Rhizobium etli


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This regulatory sequence was amplified by PCR using the genome of R. etli strain CFN42 as template DNA. The oligos used to amplify the promoter region from the mentioned genome are the following:

  • Upper primer: this primer contains the prefix sequence along with the first 25 nt of the promoter region.
    • CGGAATTCGCGGCCGCTTCTAGAGCGACACCGTCTGTCGGCTTTGTCTG


  • Lower primer: this primer contains the suffix sequence along with the last 24 nt of the promoter region (they do not include the RBS we used in our design).
    • AACTGCAGCGGCCGCTACTAGTATCTTTCGTTCCATCCATCGCTGAG


Source

This is the upstream regulatory sequence of the nifHa gene in Rhizobium etli, which encodes a nitrogenase reductase. The R. etli nifHa gene is located on the symbiotic plasmid p42d, which is a circular molecule of 371,255 bp that has the repABC type of replicator. There are three copies of the nitrogenase reductase genes in this plasmid: nifHa, nifHb and nifHc. The nifHa and nifHb copies are transcriptionally coupled to nitrogenase structural genes, forming the nifHDK operons [2]. The nifHc copy is linked to a truncated nifD homolog. A remarkable difference among them is the absence of canonical NifA binding sites upstream nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb.

References

1. Beat Thöny, Hauke Hennecke. 1989. The -24/-12 promoter comes of age. Microbiology Reviews 63, 341-358.

2. Brenda Valderrama, Araceli Dávalos, Lourdes Girard, Enrique Morett. 1996. Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated NifH genes. Journal of Bacteorology, 178, 3119-3126.

3. Emmanuel Salazar, Javier Díaz. 2010. Characterization of the NifH-RpoN regulon in Rhizobium etli in free life and in symbiosis with Phaseolus Vulgaris. Applied and Enviromental Microbiology, 76, 4510-4520.

4. Víctor González, Patricia Bustos, Miguel A. Ramirez-Romero. 2003. The mosaic structure of the symbiotic plasmid of Rhizobium etli CFN42 and its relation to other symbiotic genome compartments. Genome Biol. 4:R36.

5. Ray Dixon, Daniel Kahn (2004) Genetic regulation of biological nitrogen fixation. Nature Reviews 2, 621-631.