Coding

Part:BBa_K5306003

Designed by: Iulia Beres   Group: iGEM24_MSP-Maastricht   (2024-09-26)

nasC Electron Transporter Protein

This basic part encodes the nasC, an NADH dependent protein subunit of the nitrate reductase in the ANRA pathway (Lin et al., 1994).

Assimilatory nitrate reduction to ammonium (ANRA) in bacteria occurs through 3 main steps: (1) Nitrate uptake from the extracellular space by a transporter, (2) Conversion of nitrate to nitrite by the nitrate reductase enzyme and (3) Reduction to ammonium of the nitrite by nitrite reductase (Lin & Stewart, 1997).

Usage and Biology

Assimilatory nitrate reductase catalyzes the reduction of nitrate to nitrite once it has been trasported into the intracellular space. It is composed of two subunits: one α-subunit and one β-subunit (nasC), in addition to one molybdenum-molybdopterin (Mo-MPT) prosthetic group (Coelho & Romão, 2015).

nasC is the electron transfer subunit of nitrate reductase. This polypeptide facilitates the transfer of electrons from NADH to the catalytic subunit (nasA) of the enzyme (Lin et al., 1994). The coding sequence has been derived from the organims Klebsiella Oxytoca M5al by Wu & Stewart(1998) and has undergone codon optimization for Vibrio natriegens .

Considerations

To ensure proper enzymatic activity of nitrate reductase, our team suggests inducing the expression of nasC together with the other subunit nasA (BBa_5306005). If you want to introduce the whole nitrate assimilation pathway as our team did, you can find the whole operon sequence that contains all the coding sequences that you need in our composite part’s page (BBa_5306006) or access each one individually from this list:


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 743
    Illegal XbaI site found at 667
    Illegal XbaI site found at 1011
    Illegal XbaI site found at 1168
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 743
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 743
    Illegal BamHI site found at 1005
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 743
    Illegal XbaI site found at 667
    Illegal XbaI site found at 1011
    Illegal XbaI site found at 1168
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 743
    Illegal XbaI site found at 667
    Illegal XbaI site found at 1011
    Illegal XbaI site found at 1168
    Illegal SpeI site found at 303
    Illegal SpeI site found at 997
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  1. Lee, H. H., Ostrov, N., Wong, B. G., Gold, M. A., Khalil, A. S., & Church, G. M. (2019). Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. *Nature Microbiology*, *4*(7), 1105–1113. https://doi.org/10.1038/s41564-019-0423-8
  2. Lin, J. T., Goldman, B. S., & Stewart, V. (1993). Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al. *Journal of Bacteriology*, *175*(8), 2370–2378. https://doi.org/10.1128/jb.175.8.2370-2378.1993
  3. Lin, J. T., Goldman, B. S., & Stewart, V. (1994). The nasFEDCBA operon for nitrate and nitrite assimilation in Klebsiella pneumoniae M5al. *Journal of Bacteriology*, *176*(9), 2551–2559. https://doi.org/10.1128/jb.176.9.2551-2559.1994
  4. Moreno-Vivián, C., & Flores, E. (2007, January 1). *Chapter 17 - Nitrate Assimilation in Bacteria* (H. Bothe, S. J. Ferguson, & W. E. Newton, Eds.). ScienceDirect; Elsevier. https://www.sciencedirect.com/science/article/abs/pii/B9780444528575500187?via%3Dihub
  5. Wu, Q., & Stewart, V. (1998). NasFED Proteins Mediate Assimilatory Nitrate and Nitrite Transport in Klebsiella oxytoca (pneumoniae) M5al. *Journal of Bacteriology*, *180*(5), 1311–1322. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107022/
  6. Coelho, C. and Romão, M.J. (2015), Structural and mechanistic insights on nitrate reductases. Protein Science, 24: 1901-1911. https://doi.org/10.1002/pro.2801
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