Composite

Part:BBa_K4115043

Designed by: Kaijun Wang   Group: iGEM22_ShanghaiTech_China   (2022-09-30)


lac UV5 promoter-nifA-PnifH-sfGFP-B0015

lac UV5 promoter-nifA-PnifH-sfGFP-B0015
Function Gene regulator
Use in A.caulinodans ORS571
RFC standard None
Backbone pBBR1
Submitted by ShanghaiTech_China

This composite part is used to regulate PnifH (BBa_K4115014) by nifA protein. In the original A.caulinodans genome, the expression of the downstream PnifH can make nitrogen-fixing gene clusters functional. Thus, we transformed this composite part into the backbone of the pBBR1 shuttle plasmid and transferred it into A.caulinodans ORS571 to verify whether the nifA protein could regulate PnifH by observing the expression of the downstream superfolder GFP (BBa_K4115000).


Usage and Biology

The nifA gene (BBa_K4115002) is regulated by the lac UV5 promoter (BBa_K4115012). When there is no IPTG induction, the lac UV5 promoter is inhibited and the nifA cannot be expressed. When induced by IPTG, the lac UV5 promoter is activated to express the downstream nifA. The expressed nifA protein then regulates PnifH to activate the downstream sfGFP expression. (Figure 1)
Thus, we can conclude whether the nifA-PnifH regulating system is functional by observing the fluorescence intensity of sfGFP.

Figure 1: Gene pathway map of the nifA-PnifH regulating system.
















Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 82
    Illegal PstI site found at 223
    Illegal PstI site found at 1087
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 223
    Illegal PstI site found at 1087
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 724
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 82
    Illegal PstI site found at 223
    Illegal PstI site found at 1087
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 82
    Illegal PstI site found at 223
    Illegal PstI site found at 1087
    Illegal NgoMIV site found at 1344
    Illegal NgoMIV site found at 1539
    Illegal NgoMIV site found at 1614
    Illegal AgeI site found at 973
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1182
    Illegal BsaI.rc site found at 2101
    Illegal SapI site found at 405
    Illegal SapI site found at 1026

Reference

[1] Ryu, MH., Zhang, J., Toth, T. et al. Control of nitrogen fixation in bacteria that associate with cereals. Nat Microbiol 5, 314–330 (2020). doi: https://doi.org/10.1038/s41564-019-0631-2

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