Basic Parts

After knowing which enzyme parts, the active parts of four different reductases, and from which bacterium we were going to use in our project, we had to find the exact gene sequences for those enzymatic parts to design appropriate primers for the gene amplification via PCR. We found the respective genes of Paracoccus denitrificans on KEGG and designed our primers accordingly.

In the following section we present the gene segments and primers that were also added as basic parts to the registry.

napA

picture napA — Figure 1. Nitrate reductase (NapA) in green in complex with cytochrome in cyan (napB) showed as a cartoon representation and surface of the protein complex. The image was taken using the pdb accession 3ML1 in Pymol.

napA is the large subunit of the nitrate reductase enzyme besides napB and napC which converts nitrate (NO₃^-) to nitrite (NO₂^-). It contains a molybdopterin cofactor catalytic subunit and a 4Fe-4S cluster [1]. The exact gene sequence for this subunit in Paracoccus denitrificans was taken from KEGG: https://www.genome.jp/entry/pde:Pden_4721.

The napA primer was designed according to this sequence. However, the agarose gels done after the PCR revealed at first no band at the expected fragment size. After adjusting (conditions) two bands were visible around the expected size (2496 bp).

Part names

BBa_K4816001 - primer forward
BBa_K4816002 - primer reverse
BBa_K4816009 - napA

nirS

picture nirS — Figure 2. Nitrite reductase (NirS) in cyan showed as a cartoon representation and surface. The image was taken using the pdb accession 6TPO in Pymol.

The cytochrome cd1-nitrite reductase is the nirS subunit of the dissimilatory nitrite reductase, which catalyzes the reaction from nitrite (NO₂^-) to nitric oxide (NO). Another subunit of this reductase, which was obliged in our project includes a copper nitrite reductase, nirK [1]. The gene sequence for subunit nirS in P. denitrificans was taken from KEGG: https://www.genome.jp/entry/pde:Pden_2487.

The nirS primer was designed according to this sequence. The agarose gels after PCR amplification revealed a band at the expected size (1791 bp).

Part names

BBa_K4816003 - primer forward
BBa_K4816004 - primer reverse
BBa_K4816011 - nirS

norB

picture norB — Figure 3. Nitric oxide reductase (NorB) in orange and NorC in purple showed as a cartoon representation and surface. The image was taken using the pdb accession 5GUW in Pymol.

norB is a cytochrome b subunit with 12 transmembrane regions, which together with the other subunit norC, forms the enzyme nitric oxide reductase that transforms nitric oxide (NO) to nitrous oxide (N₂O) [1]. The P. denitrificans gene for this subunit was taken from KEGG: https://www.genome.jp/entry/pde:Pden_2483.

The norB primer was designed according to this sequence. The agarose gels after PCR amplification revealed a band at the expected size (1389 bp).

Part names

BBa_K4816005 - primer forward
BBa_K4816008 - primer reverse
BBa_K4816010 - norB

nosZ

picture nosZ — Figure 4. Nitrous oxide reductase (NosZ) in light blue showed as a cartoon representation and surface. The image was taken using the pdb accession 1FWX in Pymol.

The nosZ gene encodes the catalytic subunit of the nitrous oxide reductase, which performs the final step in nitrate reduction by catalyzing the reaction from nitrous oxide (N₂O) to gaseous nitrogen (N2) [1]. The nosZ gene from P. denitrificans was taken from KEGG: https://www.genome.jp/entry/pde:Pden_4219.

The nosZ primer was designed according to this sequence, and the agarose gels after PCR amplification revealed a band at the expected size (1959 bp).

Part names

BBa_K4816006 - primer forward
BBa_K4816007 - primer reverse
BBa_K4816012 - nosZ

References

Philippot, L. (2002). Denitrifying genes in bacterial and Archaeal genomes. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1577(3), 355–376.
https://doi.org/10.1016/s0167-4781(02)00420-7