Difference between revisions of "Part:BBa K4385013"
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The digestion and agarose gel electrophoresis of PZraP-GFP was performed by a standard protocol (Fig. 2A). The fluorescence intensity at different concentration of zinc ions was measured. As shown in Fig. 2B, the expression intensity of PZraP increased with the increase of zinc concentration. However, at 4 hrs of induction, the growth trend of fluorescence intensity began to slow down, indicating that although the PZraP would continue to be functional, the expression efficiency began to decline. | The digestion and agarose gel electrophoresis of PZraP-GFP was performed by a standard protocol (Fig. 2A). The fluorescence intensity at different concentration of zinc ions was measured. As shown in Fig. 2B, the expression intensity of PZraP increased with the increase of zinc concentration. However, at 4 hrs of induction, the growth trend of fluorescence intensity began to slow down, indicating that although the PZraP would continue to be functional, the expression efficiency began to decline. | ||
| − | [[Image: | + | [[Image: Zn_sensor2.png|center|frame|100px|<b>Figure 2.Zn2+ induction of PZraP promoter.](A) Digestion and gel electrophoresis of PzraP-GFP. (B) Fluorescence intensity of GFP under different concentration of Zn2+. The design construct was transformed into E. coli XL1-Blue. The Zn2+ was added when the OD600 value was reached 0.5. The fluorescence intensity was detected at indicated time and normalized by the OD600 value. *, P < 0.05 from control using Student’s t test. ]<br><br> |
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Revision as of 02:54, 12 October 2022
Zinc sensor
The zinc sensor part consists of the PzraP promoter, ZraSR two-component membrane associated sensor kinase system and GFP.
Usage and Biology
ZraSR consists of histidine kinase ZraS, cytoplasmic transcription factor ZraR and periplasmic accessory protein ZraP, responsible for sensing the presence of external Zn2+. The ZraSR system regulates the expression of zraP, which encodes a periplasmic zinc-binding protein, in response to high concentrations of extracellular zinc ions; phosphorylated ZraR triggers divergent transcription of zraP and zraSR.
Characterization
PZraP is a Zn2+sensing promoter which only start transcription in the presence of Zn2+. To investigate the sensitivity of PZraP, a GFP was connected as a reporter gene.
The digestion and agarose gel electrophoresis of PZraP-GFP was performed by a standard protocol (Fig. 2A). The fluorescence intensity at different concentration of zinc ions was measured. As shown in Fig. 2B, the expression intensity of PZraP increased with the increase of zinc concentration. However, at 4 hrs of induction, the growth trend of fluorescence intensity began to slow down, indicating that although the PZraP would continue to be functional, the expression efficiency began to decline.
[[Image: Zn_sensor2.png|center|frame|100px|Figure 2.Zn2+ induction of PZraP promoter.](A) Digestion and gel electrophoresis of PzraP-GFP. (B) Fluorescence intensity of GFP under different concentration of Zn2+. The design construct was transformed into E. coli XL1-Blue. The Zn2+ was added when the OD600 value was reached 0.5. The fluorescence intensity was detected at indicated time and normalized by the OD600 value. *, P < 0.05 from control using Student’s t test. ]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 907