Part:BBa_K5078001

NosZ from Dechloromonas denitrificans

Usage and Biology

NosZ-D.deniti is the Nitrous Oxide Reductase gene from Dechloromonas denitrificans, codon optimized for expression in Chlamydomonas reinhardtii. Nitrous oxide reductase is responsible for the reduction of nitrous oxide (N₂O) into water and dinitrogen (N₂) by acting as the catalysis of a copper-dependent two-electron reduction of N₂O [1]. This ensures that a bacterial host will not have a truncated nitrogen pathway, and will put harmless N₂ in the atmosphere instead of the greenhouse gas N₂O. Additionally, we hope this will cause the transformed host to uptake more nitrogen from its environment. Note NosZ-Denit is similar to NosZ-P.Stuzeri (BBa_K5078000).

Figure 1. NosZ-D.denti in a level 0 plasmid for later golden gate assembly.

Verification of Nosz-P.stu

Successful transformation of pL0-D.den into host bacterium can be determined by a restriction digest with the restriction enzyme BbsI-HF, with expected band lengths being 2292bp and 2088bp. Additionally, bacterial colonies should appear white in the present X-gal.

Figure 2. pL0-NosZ-D.denit diagnostic digest using BbsI on a 1% agarose gel. The restriction digest indicated that the two colonies taken from both cultures 1 and 2 all have pL0-NosZ-D.denit.

Structure simulation

Figure 3. Structure prediction of nitrous oxide reductase by NosZ-D.denit using AlphaFold. The structure has two identical 65.8kDa subunits that each contain 6 copper atoms [1]. The dark blue is high confidence of the structure and red is low confidence.

References

[1] Wan, S., Johnson, A. M., & Altosaar, I. (2012). Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes. Ecology and evolution, 2(2), 286–297. https://doi.org/10.1002/ece3.74

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 777
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 777
  Illegal NotI site found at 2158
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1301
  Illegal BamHI site found at 227
  Illegal BamHI site found at 965
  Illegal XhoI site found at 580
  Illegal XhoI site found at 697
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 777
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 777
  Illegal AgeI site found at 160
  Illegal AgeI site found at 1403
  
• 1000
  COMPATIBLE WITH RFC[1000]