Part:BBa_K5078006

PL1 Transcriptional Unit for NosZ

pL1-NosZ-D.denit (pL1-D.den) is the combination of the Psad Promoter+5' UTR (BBa_K3002001), NosZ D. denitrificans, the nitrous oxide reductase gene from Dechloromonas denitrificans 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-D.den is the nitrogen half of our completed nutrient uptake plasmid (BBa_K5078009). pL1-D.den is similar to our other nitrogen uptake plasmid pL1- NosZ-P.stuzeri (BBa_K5078005) in that they are both 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). 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]. Additionally, 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. The difference between them is that pL1-D.den has a higher affinity for N₂O than pL1-NosZ-P-stuzeri [2]. We chose to use both, in different final level 2 plasmid builds, as we were unsure how they would function in Chlamydomonas reinhardtii.

Figure 1| Plasmid diagram of pL1-NosZ-D.denit using benchling for modeling

Plasmid verification

Successful transformation of pL1-D.den into host bacterium can be determined by a restriction digest with the restriction enzyme BamHI-HF, with expected band lengths being 5500bp, 2300bp, and 700bp. Additionally, bacterial colonies should appear white in the present X-gal, and fluoresce red due to the mCherry gene.

Figure 2| pL1-NosZ-D.denit diagnostic digest using BamHI-HF on a 8% agarose gel (5.23.24). The restriction digest indicated promising results at 9, 10, 12, 13, and 14.

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

[2] Yoon, S., Nissen, S., Park, D., Sanford, R. A., & Löffler, F. E. (2016). Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ. Applied and environmental microbiology, 82(13), 3793–3800. https://doi.org/10.1128/AEM.00409-16

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2941
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2941
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2941
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2941
  
• 1000
  COMPATIBLE WITH RFC[1000]