Part:BBa_K4259002

NapA

napA is a periplasmic nitrate reductase and catalyzes the transformation of nitrate to nitrite in prokaryotes¹. This sequence comes from the metal resistant bacteria Cupriavidus metallidurans (C. metallidurans).

Usage and Biology

The periplasmic nitrate reductases catalyze the transformation of nitrate (NO3-) to nitrite (NO2-), meaning that NapA has a high affinity for nitrate.¹ In previous studies it was seen that it has emerged as a strong candidate for the production of silver nanoparticles (AgNP) in bacteria, especially in a nitrate-rich medium. Nitrate-rich media are known inducers of nitrate reductase. It was shown that AgNP absorbance was nearly tripled in this medium compared to a nitrate-poor media. Additionally, silver resistant E. coli strains were exposed to Ag+ and found to produce AgNPs.² These were promising results to use this gene in the Binanox project to produce bimetallic silver and gold nanoparticles.

Design

In our project we used napA from the bacterium Cupriavidus metallidurans, as it is metal resistant and a suitable candidate for NP formation.² The sequence was codon-optimized for E. coli. Then two overhang sequences with the restriction enzymes BamHI and NdeI were designed and added to the sequence. This part is therefore compatible with vectors that have those two restriction sites. In the Binanox project, the vector pET-16b was used.

References

1. Sparacino-Watkins, C., Stolz, J. F. & Basu, P. Nitrate and periplasmic nitrate reductases. Chemical Society Reviews vol. 43 676–706 Preprint at https://doi.org/10.1039/c3cs60249d (2014).

2. Chen, A., Keitz, B. K., & Contreras, L. M. (2018). Biological links between nanoparticle biosynthesis and stress responses in bacteria. Mexican journal of biotechnology, 3(4), 44-69.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 2517
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 519
  Illegal NgoMIV site found at 1310
  Illegal NgoMIV site found at 1754
  
• 1000
  COMPATIBLE WITH RFC[1000]