Part:BBa_K4816009
Nitrate Reductase (napA) from Paracoccus denitrificans PD1222
Biological Background
Denitrification is a vital component of the nitrogen cycle, a biological process that converts nitrates and nitrites into nitrogen gas, ultimately returning nitrogen to the atmosphere. This cycle is primarily orchestrated by various types of bacteria, and denitrification can occur under two distinct conditions: aerobic and anaerobic.
Aerobic denitrification, in particular, is facilitated by specific bacteria that contain a set of four critical enzymes located in the periplasm. These enzymes play pivotal roles in the denitrification process:
- Nitrate Reductase (NapA) BBa_K4816009
- Nitrite Reductase (NirS) BBa_K4816011
- Nitric Oxide Reductase (NorB) BBa_K4816010
- Nitrous Oxide Reductase (NosZ) BBa_K4816012
The Denitrifying cycle consists of a series of reactions that lead to the production of nitrogen gas, the biochemical pathway is a four-step process where first nitrate (NO3-) is reduced to nitrite (NO2-) by nitrogen reductase, the catalytic active part expressed by the gene napA, then nitrite is further reduced to nitric oxide (NO) by nitrite reductase (nirS), nitric oxide is reduced to nitrous oxide (N2O), which is a potent greenhouse gas, by the enzyme nitric oxide reductase (norB), finally the limiting step of the pathway is the reduction of nitrous oxide into nitrogen gas (N2) by the enzyme nitrous oxide reductase (nosZ). As shown in equation 1.
Equation 1: Denitrifying cycle shown as a serie of biochemical equilibrium reactions.
One notable bacterium frequently employed in denitrification studies is Paracoccus denitrificans. The preference for Paracoccus denitrificans over organisms like Pseudomonas aeruginosa is often due to the latter's pathogenic nature. Paracoccus denitrificans, on the other hand, is non-pathogenic and can serve as a safer model for denitrification research.
Additionally, Paracoccus denitrificans holds further significance in the context of synthetic biology. This bacterium is more closely related to cyanobacteria, photosynthetic organisms capable of harnessing solar energy to convert carbon dioxide into organic compounds. This connection suggests the potential for future applications in synthetic biology, where Paracoccus denitrificans enzyme may be engineered to enhance environmental sustainability and resource utilization using Cyanobacteria chasis.
To get more information about the project visit the wiki iGEM Bonn-Rheinbach 2023: NitraNix
Gene Isolation
Paracoccus denitrificans PD1222 gene encoding for napA part of the plasmid (NCBI ID: CP000491.1) located between base pairs (207310 and 209805). NapA is the active subunit of the nitrate reductase. This modified gene contains two unique restriction sites, NdeI flanking the 5' terminus and XhoI flanking the 3' terminus. Using the primers foward BBa_K4816001 and reversed BBa_K4816002 to obtain the gene of interest by PCR from Paracoccus denitrificans PD1222 plasmid DNA. The restriction site addition allows the insertion into different vectors. In this case, we use them to insert in the plasmid pet 23 a (+) to further protein expression and isolation, because the plasmid add a His-Tag to the C-terminus of the protein. Important to notice the deletion of the stop codon.
Usage and Biology
NapA was obtained by PCR amplification of genomic DNA at 45°c obtaining a prominent band at nearly 2.5kb. A repeat was made in order to corroborate the presence of NapA inside the vector of interest pet 23 a+ to validate the right insertion. Note the lower bands could be unspecific binding of the primer due to low temperature or incomplete PCR reactions.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1003
Illegal PstI site found at 127 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1003
Illegal PstI site found at 127 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1003
Illegal BglII site found at 1208
Illegal BglII site found at 1352
Illegal BamHI site found at 534
Illegal BamHI site found at 969
Illegal BamHI site found at 1451
Illegal BamHI site found at 2448
Illegal XhoI site found at 2506 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1003
Illegal PstI site found at 127 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1003
Illegal PstI site found at 127
Illegal NgoMIV site found at 1302
Illegal NgoMIV site found at 2065
Illegal NgoMIV site found at 2130 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1041
//cds/enzyme
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