Difference between revisions of "Part:BBa K4115002"
 
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The nifA protein encoded by the nifA sequence is a transcriptional activator that can activate the transcription of the downstream nifH promoter under the influence of cytosolic nitrogen sources or oxygen, and is a key activator of nitrogen-fixing gene clusters. The nitrogen fixation gene can be regulated by knocking out the native nifA sequence and replacing it with the nifA sequence added with regulatory elements.
 
The nifA protein encoded by the nifA sequence is a transcriptional activator that can activate the transcription of the downstream nifH promoter under the influence of cytosolic nitrogen sources or oxygen, and is a key activator of nitrogen-fixing gene clusters. The nitrogen fixation gene can be regulated by knocking out the native nifA sequence and replacing it with the nifA sequence added with regulatory elements.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4115002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4115002 SequenceAndFeatures</partinfo>
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===Usage and Biology===
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The A. caulinodans nif cluster is difficult to engineer because it is large (64 kb total, 76 genes), distributed across multiple loci and has a complex regulatory network.
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Fortunately, studies have shown that the expression level of nitrogen fixation genes can be efficiently regulated by regulating the expression of a single gene corresponding to nifA. [1]<br>Therefore, we followed the regulatory scheme pointed out in the literature, knocked out the original nifA gene of nitrogen-fixing bacteria regulated by ammonium root and replaced it with the nifA coding sequence regulated by other artificially designed parts. The expressed protein then activates the downstream receiving part PnifH<partinfo>BBa_K4115014</partinfo>, which in turn regulates the nif gene cluster (nitrogen-fixing gene cluster).
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[[File:Control of nitrogen fixation in A caulinodans ORS571.jpg|1000px|thumb|centre|'''Figure 1:''' a, Schematic of the controller carried on the plasmid pMR124 (top left). NifA and RpoN co-induce the expression of three sites in the genome ((i)–(iii), top right and bottom; identified by consensus NifA binding sequences). b, The levels of expression induced by the nifH promoter were evaluated using a fluorescent reporter. NifA and RpoN were complemented individually or in combination in the A. caulinodans ΔnifA, where the genomic rpoN remains intact. c, Response function for the induction of the nifH promoter by the controller under nitrogen-fixing conditions. d, Nitrogenase activity of wild-type A. caulinodans ORS571 compared with ΔnifA complemented with the controller plasmid and the addition of 1 mM IPTG. e, Same as in d, but the cell growth in ammonium-free agarose slopes is shown. f, Effect of the absence or presence of 10 mM ammonium chloride on the specific nitrogenase activity. NifA and RpoN expression was induced with 1 mM IPTG for A. caulinodans ΔnifA containing the controller plasmid pMR127. The asterisk indicates ethylene production at levels below the detection limit. g, Specific nitrogenase activity of the inducible version encoding the controller shown as a function of the oxygen concentration in the headspace in the presence of 1 mM IPTG. The error bars represent the s.d. from three independent experiments performed on different days. WT, wild type.The picture above is from[1]]]
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[1]Ryu, M. H., Zhang, J., Toth, T., Khokhani, D., Geddes, B. A., Mus, F., Garcia-Costas, A., Peters, J. W., Poole, P. S., Ané, J. M., & Voigt, C. A. (2020). Control of nitrogen fixation in bacteria that associate with cereals. Nature microbiology, 5(2), 314–330. https://doi.org/10.1038/s41564-019-0631-2
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4115002 parameters</partinfo>
 
<partinfo>BBa_K4115002 parameters</partinfo>
 
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Latest revision as of 10:37, 4 October 2022


NifA(nif-specific transcriptional activator)

The coding sequence of the nifA protein in the nitrogen-fixing gene cluster.


The nifA protein encoded by the nifA sequence is a transcriptional activator that can activate the transcription of the downstream nifH promoter under the influence of cytosolic nitrogen sources or oxygen, and is a key activator of nitrogen-fixing gene clusters. The nitrogen fixation gene can be regulated by knocking out the native nifA sequence and replacing it with the nifA sequence added with regulatory elements.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 136
    Illegal PstI site found at 1000
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 136
    Illegal PstI site found at 1000
    Illegal NgoMIV site found at 1257
    Illegal NgoMIV site found at 1452
    Illegal NgoMIV site found at 1527
    Illegal AgeI site found at 886
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1095
    Illegal SapI site found at 318
    Illegal SapI site found at 939

Usage and Biology

The A. caulinodans nif cluster is difficult to engineer because it is large (64 kb total, 76 genes), distributed across multiple loci and has a complex regulatory network. Fortunately, studies have shown that the expression level of nitrogen fixation genes can be efficiently regulated by regulating the expression of a single gene corresponding to nifA. [1]
Therefore, we followed the regulatory scheme pointed out in the literature, knocked out the original nifA gene of nitrogen-fixing bacteria regulated by ammonium root and replaced it with the nifA coding sequence regulated by other artificially designed parts. The expressed protein then activates the downstream receiving part PnifHBBa_K4115014, which in turn regulates the nif gene cluster (nitrogen-fixing gene cluster).

Figure 1: a, Schematic of the controller carried on the plasmid pMR124 (top left). NifA and RpoN co-induce the expression of three sites in the genome ((i)–(iii), top right and bottom; identified by consensus NifA binding sequences). b, The levels of expression induced by the nifH promoter were evaluated using a fluorescent reporter. NifA and RpoN were complemented individually or in combination in the A. caulinodans ΔnifA, where the genomic rpoN remains intact. c, Response function for the induction of the nifH promoter by the controller under nitrogen-fixing conditions. d, Nitrogenase activity of wild-type A. caulinodans ORS571 compared with ΔnifA complemented with the controller plasmid and the addition of 1 mM IPTG. e, Same as in d, but the cell growth in ammonium-free agarose slopes is shown. f, Effect of the absence or presence of 10 mM ammonium chloride on the specific nitrogenase activity. NifA and RpoN expression was induced with 1 mM IPTG for A. caulinodans ΔnifA containing the controller plasmid pMR127. The asterisk indicates ethylene production at levels below the detection limit. g, Specific nitrogenase activity of the inducible version encoding the controller shown as a function of the oxygen concentration in the headspace in the presence of 1 mM IPTG. The error bars represent the s.d. from three independent experiments performed on different days. WT, wild type.The picture above is from[1]

[1]Ryu, M. H., Zhang, J., Toth, T., Khokhani, D., Geddes, B. A., Mus, F., Garcia-Costas, A., Peters, J. W., Poole, P. S., Ané, J. M., & Voigt, C. A. (2020). Control of nitrogen fixation in bacteria that associate with cereals. Nature microbiology, 5(2), 314–330. https://doi.org/10.1038/s41564-019-0631-2