Part:BBa_K5078005

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), 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 cause the transformed C. reinhardtii to uptake more nitrogen from their environment in the final L2 construct.

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 digest 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 fluoresce red due to the mCherry gene.

Figure 2| pL1-NosZ-P.stuzeri diagnostic digest using BbsI on a 1% 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]