Difference between revisions of "Part:BBa K5078005"

 
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This is a level 1 plasmid build of nosZ P. stutzeri. Having added a PSAD promoter, PSAD terminator, and the fluorescence gene mcherry.
 
This is a level 1 plasmid build of nosZ P. stutzeri. Having added a PSAD promoter, PSAD terminator, and the fluorescence gene mcherry.
  
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===Usage and Biology===
 
===Usage and Biology===
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pL1-NosZ-P.stutzeri (pL1-P.stu) is the combination of the Psad Promoter+5' UTR (BBa_K3002001), the NosZ-P.stutzeri the nitrous oxide reductase gene from Pseudomonas stutzerii codon optimized for expression in Chlamydomonas reinhardtii (BBa_K5078000), the N-terminator sequence of the fluorescent reporter gene mCherry (BBa_K4770013), and the Psad Terminator (BBa_K3002002). pL1-P.stu is the nitrogen half of our completed nutrient uptake plasmid (BBa_K5078009). pL1-P.stu is responsible for altering the nitrogen pathway of a host bacterium by forcing the host bacterium to undergo the final step of denitrification, the reduction of nitrous oxide (N₂O) into dinitrogen (N₂). pL1-P.stu does this by encoding for nitrous oxide reductase, which will act as the catalysis of a copper-dependent two electron reduction of N₂O into water and N₂ [1]. This has the benefit of preventing the greenhouse gas N₂O from entering the atmosphere and theoretically should causes the host bacterium to uptake more nitrogen from their environment.
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<html><div style="text-align: center;">
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<img src="https://static.igem.wiki/teams/5078/plasmid-pictures/nosz-pstu-l1-picture.webp" width="400" height="auto"/><br>
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Figure 1| Plasmid diagram of pL1-NosZ-P.stutzeri using benchling for modeling.</div></html>
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==Plasmid verification==
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Successful transformation of pL1-P.stu into host bacterium can be determined by a restriction digestion with the restriction enzyme BbsI, with expected band lengths at 4352bp and 4160bp. Additionally bacterial colonies should appear white in the present X-gal, and fluoresces red under UV light due to the mCherry gene.
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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-l1.webp" width="400" height="auto"/><br>
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Figure 2| pL1-NosZ-P.stuzeri diagnostic digest using BbsI on a 8% agarose gel (5.22.24). The restriction digest indicated two good bands on P. stutzeri #2.
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</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
  
 
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Revision as of 19:05, 28 September 2024


pL1 nosZ P. stutzeri

This is a level 1 plasmid build of nosZ P. stutzeri. Having added a PSAD promoter, PSAD terminator, and the fluorescence gene mcherry.

Usage and Biology

pL1-NosZ-P.stutzeri (pL1-P.stu) is the combination of the Psad Promoter+5' UTR (BBa_K3002001), the NosZ-P.stutzeri the nitrous oxide reductase gene from Pseudomonas stutzerii codon optimized for expression in Chlamydomonas reinhardtii (BBa_K5078000), the N-terminator sequence of the fluorescent reporter gene mCherry (BBa_K4770013), and the Psad Terminator (BBa_K3002002). pL1-P.stu is the nitrogen half of our completed nutrient uptake plasmid (BBa_K5078009). pL1-P.stu is responsible for altering the nitrogen pathway of a host bacterium by forcing the host bacterium to undergo the final step of denitrification, the reduction of nitrous oxide (N₂O) into dinitrogen (N₂). pL1-P.stu does this by encoding for nitrous oxide reductase, which will act as the catalysis of a copper-dependent two electron reduction of N₂O into water and N₂ [1]. This has the benefit of preventing the greenhouse gas N₂O from entering the atmosphere and theoretically should causes the host bacterium to uptake more nitrogen from their environment.


Figure 1| Plasmid diagram of pL1-NosZ-P.stutzeri using benchling for modeling.

Plasmid verification

Successful transformation of pL1-P.stu into host bacterium can be determined by a restriction digestion with the restriction enzyme BbsI, with expected band lengths at 4352bp and 4160bp. Additionally bacterial colonies should appear white in the present X-gal, and fluoresces red under UV light due to the mCherry gene.


Figure 2| pL1-NosZ-P.stuzeri diagnostic digest using BbsI on a 8% agarose gel (5.22.24). The restriction digest indicated two good bands on P. stutzeri #2.

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 1356
    Illegal PstI site found at 3081
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2118
    Illegal PstI site found at 1356
    Illegal PstI site found at 3081
    Illegal NotI site found at 960
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2335
    Illegal BamHI site found at 1282
    Illegal XhoI site found at 849
    Illegal XhoI site found at 2403
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1356
    Illegal PstI site found at 3081
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1356
    Illegal PstI site found at 3081
    Illegal NgoMIV site found at 1890
  • 1000
    COMPATIBLE WITH RFC[1000]