Difference between revisions of "Part:BBa K5078000"

 
 
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<partinfo>BBa_K5078000 short</partinfo>
 
<partinfo>BBa_K5078000 short</partinfo>
  
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Promoter and 5' UTR for high level expression in Chlamydomonas reinhardtii
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This is the Nitrous Oxide Reductase gene from Pseudomonas stutzeri 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.
  
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<html><div style="text-align: center;">
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===Usage and Biology===
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<img src="https://static.igem.wiki/teams/5078/plasmid-pictures/nosz-p-stutzerii-l0-plasmid-picture.webp" width="400" height="auto"/><br>
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Figure 1. NosZ P.stutzeri in a level 0 plasmid for later golden gate assembly.</div></html>
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===Verification of nosZ P. stu===
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Successful transformation of pL0-P.stu into host bacterium can be determined by a restriction digest with the restriction enzyme BbsI, with the molecular weights being 1914bp and 2088bp. Additionally, bacterial colonies should appear white in the present X-gal.
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<html><div style="text-align: center;">
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<img src="https://static.igem.wiki/teams/5078/experiments/digest-of-pstu-and-ddenit-l0.png" width="400" height="auto"/><br>
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Figure 2. pL0-NosZ-P.stuzeri diagnostic digest using BbsI on a 8% agarose gel. The restriction digest indicated that the two colonies taken from both cultures 1 and 2 all have pL0-NosZ-P.stuzeri.</div></html>
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===Structure simulation===
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<html><div style="text-align: center;">
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<img src="https://static.igem.wiki/teams/5078/modeling/p-stu-nosz-folding-sphere.webp" width="400" height="auto"/><br>
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Figure 3. Structure prediction of nitrous oxide reductase. The structure has two identical 65.8kDa subunits that each contain 6 copper atoms</div></html>
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====References====
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[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<br>
  
 
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Latest revision as of 00:24, 2 October 2024


Nos Z gene from P. stutzeri

This is the Nitrous Oxide Reductase gene from Pseudomonas stutzeri 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.


Figure 1. NosZ P.stutzeri in a level 0 plasmid for later golden gate assembly.

Verification of nosZ P. stu

Successful transformation of pL0-P.stu into host bacterium can be determined by a restriction digest with the restriction enzyme BbsI, with the molecular weights being 1914bp and 2088bp. Additionally, bacterial colonies should appear white in the present X-gal.


Figure 2. pL0-NosZ-P.stuzeri diagnostic digest using BbsI on a 8% agarose gel. The restriction digest indicated that the two colonies taken from both cultures 1 and 2 all have pL0-NosZ-P.stuzeri.

Structure simulation


Figure 3. Structure prediction of nitrous oxide reductase. The structure has two identical 65.8kDa subunits that each contain 6 copper atoms

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 541
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1303
    Illegal PstI site found at 541
    Illegal NotI site found at 145
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1520
    Illegal BamHI site found at 467
    Illegal XhoI site found at 34
    Illegal XhoI site found at 1588
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 541
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 541
    Illegal NgoMIV site found at 1075
  • 1000
    COMPATIBLE WITH RFC[1000]