Difference between revisions of "Part:BBa K5306001"
 
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<h2> Considerations </h2>
 
<h2> Considerations </h2>
  
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To ensure a proper functionality of the transporter enzyme, the sequence encoding nasE should be introduced with the other 2 subunits nasF[https://parts.igem.org/Part:BBa_K5306000 (BBa_5306000)] and nas D [https://parts.igem.org/Part:BBa_K5306002 (BBa_5306002)] as these genes has been found by Wu & Stewart (1998) to work as an operon. 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 [https://parts.igem.org/Part:BBa_K5306006 (BBa_5306006)] or access each one individually from this list:
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To ensure a proper functionality of the transporter enzyme, the sequence encoding nasE should be introduced with the other 2 subunits nasF[https://parts.igem.org/Part:BBa_K5306000 (BBa_5306000)] and nas D [https://parts.igem.org/Part:BBa_K5306002 (BBa_5306002)] as these genes have been found by Wu & Stewart (1998) to work as an operon. 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 [https://parts.igem.org/Part:BBa_K5306006 (BBa_5306006)] or access each one individually from this list:
  
 
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Latest revision as of 11:20, 1 October 2024


nasE Homodimeric Transmembrane Protein

nasE composes the membrane spanning subunit of the ABC nitrate transporter protein from the ANRA (Assimilatory Nitrate Reduction to Ammonium) pathway, responsible for allowing the selective passage of nitrate ions through the membrane while maintaing the cell’s electrochemical gradient (Lin & Stewart, 1997).

Usage and Biology

Nitrate assimilation in bacteria occurs through 3 main steps: (1) Nitrate uptake from the extracellular space by a trasporter, (2) Conversion of nitrate to nitrite by the nitrate reductase enzyme and (3) Reduction to ammonium of the nitrite by nitrite reductase.

The transporter protein is a crucial part in the ANRA pathway. It’s an ATP-binding casette (ABC) transporter enzyme (Lin & Stewart, 1997). To be able to transport the NO3- ions through the membrane, this group of transporters require energy from ATP hydroloysis. This transporter is composed of of three subunits: a periplasmic protein that binds NO3-, a transmembrane protein, and a cytoplasmic ATPase attached to the membrane (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). Its high-affinity binding of NO3- enables efficient uptake even at low external concentrations.

Image 1

The nasFED nitrate transporter complex (Wu & Stewart, 1998)

The coding sequence of nasE is derived from the organism Klebsiella Oxytoca M5al by Wu and Stewart (1998) and has undergone codon optimization for Vibrio natriegens .


Considerations

To ensure a proper functionality of the transporter enzyme, the sequence encoding nasE should be introduced with the other 2 subunits nasF(BBa_5306000) and nas D (BBa_5306002) as these genes have been found by Wu & Stewart (1998) to work as an operon. 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 566
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 566
    Illegal NheI site found at 852
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 566
    Illegal BglII site found at 385
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 566
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 566
    Illegal NgoMIV site found at 654
    Illegal AgeI site found at 667
  • 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/