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 with the final De-Slimer plasmid (BBa_K5078009). 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 2941
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 2941
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 2941
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 2941
- 1000COMPATIBLE WITH RFC[1000]
| None |