Difference between revisions of "Part:BBa K4115043"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K4115043 short</partinfo> | <partinfo>BBa_K4115043 short</partinfo> | ||
+ | {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" | ||
+ | ! colspan="2" style="background:#FFBF00;"|lac UV5 promoter-nifA-PnifH-sfGFP-B0015 | ||
+ | |- | ||
+ | |'''Function''' | ||
+ | |Gene regulator | ||
+ | |- | ||
+ | |'''Use in''' | ||
+ | |<i>A.caulinodans</i> ORS571 | ||
+ | |- | ||
+ | |'''RFC standard''' | ||
+ | |None<br> | ||
+ | |- | ||
+ | |'''Backbone''' | ||
+ | |pBBR1<br> | ||
+ | |- | ||
+ | |'''Submitted by''' | ||
+ | |[https://2022.igem.wiki/shanghaitech-china/ ShanghaiTech_China] | ||
+ | |} | ||
+ | |||
+ | This composite part is used to regulate PnifH [https://parts.igem.org/Part:BBa_K4115014 (BBa_K4115014)] by nifA protein. In the original <i>A.caulinodans</i> 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 <i>A.caulinodans</i> ORS571 to verify whether the nifA protein could regulate PnifH by observing the expression of the downstream superfolder GFP [https://parts.igem.org/Part:BBa_K4115000 (BBa_K4115000)]. | ||
− | |||
− | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
+ | |||
+ | The nifA gene [https://parts.igem.org/Part:BBa_K4115002 (BBa_K4115002)] is regulated by the lac UV5 promoter [https://parts.igem.org/Part:BBa_K4115012 (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)<br> | ||
+ | Thus, we can conclude whether the nifA-PnifH regulating system is functional by observing the fluorescence intensity of sfGFP. | ||
+ | |||
+ | [[File:NifA regulate PnifH.png|600px|thumb|left|'''Figure 1:''' Gene pathway map of the nifA-PnifH regulating system.]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> | ||
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<partinfo>BBa_K4115043 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4115043 SequenceAndFeatures</partinfo> | ||
+ | == Reference == | ||
+ | <small> | ||
+ | |||
+ | [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 | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display |
Latest revision as of 07:11, 5 October 2022
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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 82
Illegal PstI site found at 223
Illegal PstI site found at 1087 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 223
Illegal PstI site found at 1087 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 724
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 82
Illegal PstI site found at 223
Illegal PstI site found at 1087 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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