Coding

Part:BBa_K5078001

Designed by: Hagen Harmer   Group: iGEM24_UVU-Utah-2   (2024-08-01)


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]


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