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Part:BBa_K5306006

Designed by: Iulia Beres   Group: iGEM24_MSP-Maastricht   (2024-09-28)
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ANRA Pathway

This composite part encodes for the whole assimilatory nitrate reduction to ammonium (ANRA) pathway.

Usage and Biology

Nitrate assimilation in bacteria is a process by which the organism uses NO3-, and NO2-, as nitrogen sources and convert it into ammonium (NH4+). Nitrates are transported in the intracellular space by a transporter, where they are converted through nitrites into ammonium (Lin et al., 1994). Our team has selected this pathway as it provides an effective means of nitrogen acquisition and assimilation, as well as avoiding the production of harmful byproducts that can harm the microorganism.

Vibrio natriegens is a fast-growing marine organism, that thrives in a high salinity environement, with a doubling rate of less than ten minutes, making it a potential chasiss for molecular and synthetic biology applications (Weinstock et al., 2016, Stukenberg et al., 2021). Natronaut has chosen to integrate the nitrate assimilation pathway in this organism in order to adress the problem of coastal eutrophication by capturing ran-off NO3-.

Composition

This composite part encodes for the three main enzymes involved in the ANRA Pathway:

  1. Transporter (nasFED complex): This is an ABC (ATP-Binding Casette) enzyme located in the cytoplasmic membrane of the bacteria responsible for the uptake of nitrates (Wu & Stewart, 1998). It is composed of three subunits: a periplasmic binding protein that binds to extracellular NO3-, with high affinity, a transmembrane protein that allows the passage of nitrate through the lipid bilayer and a cytoplasmic ATP-binding protein (Wu & Stewart, 1998).
  2. Nitrate Reductase (nasCA complex): The nitrate reductase is an NADH-depepndent enzyme that catalyzes the conversion of NO3-, once it has entered the cell to NO2-, (Lin et al., 1993, Lin et al., 1994). It has a large catalytic subunit that contains the active site for reduction and a small NADH oxidoreductase subunit, used for transfer of electrons through the biochemical reactions(Coelho & Romão, 2015). The active site is accompanied by a molybdenum-molybdoprotein responsible for catalyzing the reduction reaction(Coelho & Romão, 2015).
  3. Nitrite Reductase (nasB): Nitrate reductase is the enzyme responsible for carrying out the last step of the pathway. Being a cytochrome enzyme, it facilitates the conversion of NO2-, into NH4+, through different heme groups, where each one converts one molecule at a time (Einsle et al., 2002). The sequence encoding nasB contains a 6x His tag to be able to locate the protein and confirm the introduction of this pathway in the organism.

Image 1
Overview of the assimilatory nitrate reductase pathway enzyme complexes (Wu & Stewart, 1998)

In our project, the converted NH4+ is subsequently assimilated for the production of single-cell proteins (SCPs) through glutamate biosynthesis. This process occurs through two pathways: glutamine synthetase (GS)-glutamate synthase (GOGAT) and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutamate dehydrogenase (Ohashi et al., 2011; van Heeswijk et al., 2013; Jiang & Jiao, 2016).


Image 2
The assimilatory nitrate to ammonium (ANRA) pathway and the Glutamate Biosynthesis for the production of single-cell proteins (SCPs) (Created with [https://www.biorender.com/ Biorender])


Operon Structure

According to Lin et al. (1994) the genes encoding the three enzyme involved in nitrate assimilation, function as an operon. They form one long molecule of mRNA that gets translated into multiple proteins. Therefore, we have chosen to integrate the whole operon sequence into the plasmid backbone under the control of only one promoter and a terminator. This allows for the coordinated expression of all three enzymes in a single messenger RNA, facilitating appropiate protein-protein interaction and communication within the pathway. By using only one promoter and terminator, we simplify the regulation of the gene expression. As it is an operon, we inserted an RBS sequence, native to V. natriegens, upstream of each gene to simulate native conditions.

Image 3
Operon structure and size (Lin et. al, 1994)

The coding sequences of the three enzymes: transporter, nitrate/nitrite reductase have been obtained from the organism Klebsiella Oxytoca M5al by Wu & Stewart (1998) and has undergone codon optimization for Vibrio natriegens .

Assembly Design & Results

To ensure a succesful expression, our team has selected a strong promoter that has been found to be native to our chasiss organism Vibrio natriegens from iGEM Groningen 2019 team (BBa_K3171171). This promoter has been reported to induce consitutive transcription of our target gene and has been documented by Tschirhart et al. (2019) to induce high gene expression levels. Moreover, Tschirhart et al. (2019) has also tested the double terminator (B0015) confirming its use in V. natriegens .

We have also selected three RBS sequences synthesized by Marburg 2018 VibriGens that are similar in strength ( BBa_K2560008, BBa_K2560010, BBa_K2560016to acquire optimal translation efficiency for the expression of our target genes. These sequences were chosen to balance protein production rates and to avoid overwhelming the cell.

Due to the sheer size of the construct (10174 bp), a vector with a low-copy number was chosen to minimize the metabolic burden on the host cell. Therefore, the backbone that was used, pSEVA261, is a plasmid with a p15A origin of replication, that has been found by Tschirhart et al. (2019) to have a high-transformation efficiency and high-maintanance.

For the purpose of this project, our team used Gibson Assembly as this method would be the most effective approach, since the fragments have a substantial length (2000 bp- 2900 bp). The construct was assembled into the pSEVA261 vector using the NEBuilder HiFi DNA Assembly by NEB and further cloned into the NEB® 10-beta Competent E. coli cells.

Characterization

To confirm the inital assembly, restriction enzymes digestion reactions were performed on the isolated plasmid. We have selected the NaeI and XhoI enzymes present in our construct and we simulated the gel electrophoresis expected results in SnapGene. After PCR we ran a 1% agarose gel and found that the results match the expectations confirming the succesful asembly of the construct.

Image 4
Restriction Enzymes Digestions (NaeI, XhoI): The results show distinctive bands at 12kb confirming the presence of the assembled plasmid (left), the expected results (right)

Considerations

Due to time constraints, our team was unable to fully characterize the enzymatic activity of the pathway in Vibrio natriegens. However, we have developed a detailed plan for future analysis that can be found on our wiki(Characterization):

  • Western-blot to locate the pathway: As we have attatched a 6x HIS tag on the nitrite reductase enzyme, facilitating its detection with anti-His antibodies and purification through nickel immobilized metal affinity chromatography (IMAC). This will allow us to analyse not only the nitrite reductase but also the localization of the pathway within the organism.

Chemical tests:

  • Griess Test: The purpose of this test is to test the enzymatic activity of the nitrate reductase protein based on the formation of a diazonium salt intermediate from the reaction between nitrate and sulfanilinic acid.
  • Indophenol Blue Test: This test is used to detect the presence of ammonia by exploiting the electrophilicity of the ammonium ion, subsequently confirming the enzymatic activity of the nitrite reductase.
From these experiments, the concentrations of the detected compounds can be measured by looking at the absorbance levels and using Beer Lambert’s Law.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 7
    Illegal suffix found in sequence at 305
    Illegal EcoRI site found at 2226
    Illegal EcoRI site found at 2638
    Illegal EcoRI site found at 4150
    Illegal XbaI site found at 3063
    Illegal XbaI site found at 4074
    Illegal XbaI site found at 4418
    Illegal XbaI site found at 4575
    Illegal XbaI site found at 5111
    Illegal XbaI site found at 6461
    Illegal SpeI site found at 3710
    Illegal SpeI site found at 4404
    Illegal PstI site found at 776
    Illegal PstI site found at 5353
    Illegal PstI site found at 6862
    Illegal PstI site found at 7798
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 7
    Illegal EcoRI site found at 2226
    Illegal EcoRI site found at 2638
    Illegal EcoRI site found at 4150
    Illegal NheI site found at 2512
    Illegal NheI site found at 3288
    Illegal NheI site found at 4958
    Illegal NheI site found at 5462
    Illegal NheI site found at 6491
    Illegal NheI site found at 8579
    Illegal SpeI site found at 306
    Illegal SpeI site found at 3710
    Illegal SpeI site found at 4404
    Illegal PstI site found at 320
    Illegal PstI site found at 776
    Illegal PstI site found at 5353
    Illegal PstI site found at 6862
    Illegal PstI site found at 7798
    Illegal NotI site found at 13
    Illegal NotI site found at 313
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 7
    Illegal EcoRI site found at 2226
    Illegal EcoRI site found at 2638
    Illegal EcoRI site found at 4150
    Illegal BglII site found at 2045
    Illegal BglII site found at 5385
    Illegal BglII site found at 6800
    Illegal BamHI site found at 4412
    Illegal XhoI site found at 2947
    Illegal XhoI site found at 9991
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 7
    Illegal suffix found in sequence at 306
    Illegal EcoRI site found at 2226
    Illegal EcoRI site found at 2638
    Illegal EcoRI site found at 4150
    Illegal XbaI site found at 3063
    Illegal XbaI site found at 4074
    Illegal XbaI site found at 4418
    Illegal XbaI site found at 4575
    Illegal XbaI site found at 5111
    Illegal XbaI site found at 6461
    Illegal SpeI site found at 3710
    Illegal SpeI site found at 4404
    Illegal PstI site found at 776
    Illegal PstI site found at 5353
    Illegal PstI site found at 6862
    Illegal PstI site found at 7798
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 7
    Illegal EcoRI site found at 2226
    Illegal EcoRI site found at 2638
    Illegal EcoRI site found at 4150
    Illegal XbaI site found at 22
    Illegal XbaI site found at 3063
    Illegal XbaI site found at 4074
    Illegal XbaI site found at 4418
    Illegal XbaI site found at 4575
    Illegal XbaI site found at 5111
    Illegal XbaI site found at 6461
    Illegal SpeI site found at 306
    Illegal SpeI site found at 3710
    Illegal SpeI site found at 4404
    Illegal PstI site found at 320
    Illegal PstI site found at 776
    Illegal PstI site found at 5353
    Illegal PstI site found at 6862
    Illegal PstI site found at 7798
    Illegal NgoMIV site found at 2314
    Illegal AgeI site found at 869
    Illegal AgeI site found at 2327
    Illegal AgeI site found at 9365
  • 1000
    COMPATIBLE WITH RFC[1000]

References

    1. Benchling. (2023). Cloud-Based Informatics Platform for Life Sciences R&D. Benchling. https://www.benchling.com/
    2. 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
    3. D3veloperSCS_SEVA. (n.d.). Find your plasmid. SEVA Plasmids - Standard European Vector Architecture. https://seva-plasmids.com/canonical-seva-plasmid-list/
    4. Einsle, O., Messerschmidt, A., Huber, R., Peter, & Neese, F. (2002). Mechanism of the Six-Electron Reduction of Nitrite to Ammonia by Cytochrome c Nitrite Reductase. Journal of the American Chemical Society, 124(39), 11737–11745. https://doi.org/10.1021/ja0206487
    5. Jiang, X., & Jiao, N. (2015). Nitrate assimilation by marine heterotrophic bacteria. Science China. Earth Sciences/Science China. Earth Sciences, 59(3), 477–483. https://doi.org/10.1007/s11430-015-5212-5
    6. 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
    7. 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
    8. 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
    9. 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
    10. Ohashi, Y., Shi, W., Takatani, N., Aichi, M., Maeda, S., Watanabe, S., Yoshikawa, H., & Omata, T. (2011). Regulation of nitrate assimilation in cyanobacteria. Journal of Experimental Botany, 62(4), 1411–1424. https://doi.org/10.1093/jxb/erq427
    11. Shetty, R. (2003, July 17). Part:BBa B0015 - parts.igem.org. Parts.igem.org. https://parts.igem.org/Part:BBa_B0015
    12. Stukenberg, D., Hensel, T., Hoff, J., Daniel, B., Inckemann, R., Tedeschi, J. N., Nousch, F., & Fritz, G. (2021). The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens. ACS Synthetic Biology, 10(8), 1904–1919. https://doi.org/10.1021/acssynbio.1c00126
    13. Team:Marburg/Part Collection - 2018.igem.org. (n.d.). 2018.Igem.org. https://2018.igem.org/Team:Marburg/Part_Collection
    14. Tschirhart, T., Shukla, V., Kelly, E. E., Schultzhaus, Z., NewRingeisen, E., Erickson, J. S., Wang, Z., Garcia, W., Curl, E., Egbert, R. G., Yeung, E., & Vora, G. J. (2019). Synthetic Biology Tools for the Fast-Growing Marine Bacterium Vibrio natriegens. ACS Synthetic Biology, 8(9), 2069–2079. https://doi.org/10.1021/acssynbio.9b00176
    15. van Heeswijk, W. C., Westerhoff, H. V., & Boogerd, F. C. (2013). Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective. Microbiology and Molecular Biology Reviews, 77(4), 628–695. https://doi.org/10.1128/mmbr.00025-13
    16. Weinstock, M. T., Hesek, E. D., Wilson, C. M., & Gibson, D. G. (2016). Vibrio natriegens as a fast-growing host for molecular biology. Nature Methods, 13(10), 849–851. https://doi.org/10.1038/nmeth.3970
    17. 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/
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