Difference between revisions of "Part:BBa K190016"

 
 
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__NOTOC__
 
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<partinfo>BBa_K190016 short</partinfo>
 
<partinfo>BBa_K190016 short</partinfo>
  
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Promoter sequence with recognition site for ZntR transcriptional regulator protein.
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Promoter sequence with recognition site for ZntR transcriptional regulator protein. ZntR activates transcription when Zn(II) is bound (1).
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=== Contribution from Squirrel-Beijing I  2024 ===
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We tested the response curve of this promoter system to various concentrations of zinc ions and performed a Hill equation fit：
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<img src="https://static.igem.wiki/teams/5081/part-registry/trancription-unit-1.png" alt="图1" style="width: 350px; margin-right: 10px;">
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<img src="https://static.igem.wiki/teams/5081/part-registry/trancription-unit-2.png" alt="图1" style="width: 350px; margin-right: 10px;">
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</div>
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Transcription Unit 1 and Transcription Unit 2 were transformed into 100 µl of T1 Chemically Competent Cells (TransGen) and plated on LB + 2% agar plates, which were incubated overnight at 37°C and stored at 4°C after growth. For the mVenusNB fluorescent kinetic assay, three single colonies of Section I or Section II strains were inoculated into 0.9 ml of LB medium with specific antibiotics in a 2-ml 96-well deep-well plate (NEST). Plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated overnight at 37°C and 1000 r.p.m. Overnight cultures were diluted 1:300 into 900 µl of LB medium with antibiotics in 2-ml 96-well deep-well plates (NEST), sealed again, and incubated at 37°C and 1000 r.p.m. After 3 hours, metal ions were added at concentrations of 0 µM Zn(II), 170 nM Zn(II), 340 nM Zn(II), 781 nM Zn(II), 15.6 µM Zn(II), 31.25 µM Zn(II), 62.5 µM Zn(II), 125 µM Zn(II), 200 µM Zn(II), 500 µM Zn(II), and 1 mM Zn(II). After mixing, 135 µl of each culture was transferred into three black 96-well cell culture plates with clear bottoms (In Vitro Scientific) to set up replicates, with an additional 15 µl of mineral oil added to prevent evaporation. Fluorescence kinetics (excitation at 515 nm, emission at 555 nm) were measured every 10 minutes overnight using a Spark® Multimode Microplate Reader. The optical density and mVenusNB fluorescence were measured over 24 hours. Initial fluorescence signals were treated as background and subtracted from each measurement. To fit the response curves for each pathway, we used the A.U. at 20 hours (mid-plateau period) and applied the Hill function, focusing on the C50 and maximum expression values.
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<img src="https://static.igem.wiki/teams/5081/part-registry/znic-od-rfu-curve.png" alt="图1" style="width: 350px; margin-right: 10px;">
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<img src="https://static.igem.wiki/teams/5081/part-registry/znic-hill.png" alt="图1" style="width: 350px; margin-right: 10px;">
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<partinfo>BBa_K190016 parameters</partinfo>
 
<partinfo>BBa_K190016 parameters</partinfo>
 
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1) C.E. Outten, F.W. Outten, T.V. O’Halloran (1999) DNA Distortion Mechanism for Transcriptional Activation by ZntR,
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a Zn(II)-responsive MerR Homologue in Escherichia coli, ''The Journal of Biological Chemistry'', Vol. 274, No. 53, Issue of December 31, pp. 37517–37524.

Latest revision as of 03:27, 2 October 2024

Zinc Promoter (ZntR regulated)

Promoter sequence with recognition site for ZntR transcriptional regulator protein. ZntR activates transcription when Zn(II) is bound (1).


Contribution from Squirrel-Beijing I 2024

We tested the response curve of this promoter system to various concentrations of zinc ions and performed a Hill equation fit：

图1 图1
Transcription Unit 1 and Transcription Unit 2 were transformed into 100 µl of T1 Chemically Competent Cells (TransGen) and plated on LB + 2% agar plates, which were incubated overnight at 37°C and stored at 4°C after growth. For the mVenusNB fluorescent kinetic assay, three single colonies of Section I or Section II strains were inoculated into 0.9 ml of LB medium with specific antibiotics in a 2-ml 96-well deep-well plate (NEST). Plates were sealed with breathable Nunc Seals (Thermo Scientific) and incubated overnight at 37°C and 1000 r.p.m. Overnight cultures were diluted 1:300 into 900 µl of LB medium with antibiotics in 2-ml 96-well deep-well plates (NEST), sealed again, and incubated at 37°C and 1000 r.p.m. After 3 hours, metal ions were added at concentrations of 0 µM Zn(II), 170 nM Zn(II), 340 nM Zn(II), 781 nM Zn(II), 15.6 µM Zn(II), 31.25 µM Zn(II), 62.5 µM Zn(II), 125 µM Zn(II), 200 µM Zn(II), 500 µM Zn(II), and 1 mM Zn(II). After mixing, 135 µl of each culture was transferred into three black 96-well cell culture plates with clear bottoms (In Vitro Scientific) to set up replicates, with an additional 15 µl of mineral oil added to prevent evaporation. Fluorescence kinetics (excitation at 515 nm, emission at 555 nm) were measured every 10 minutes overnight using a Spark® Multimode Microplate Reader. The optical density and mVenusNB fluorescence were measured over 24 hours. Initial fluorescence signals were treated as background and subtracted from each measurement. To fit the response curves for each pathway, we used the A.U. at 20 hours (mid-plateau period) and applied the Hill function, focusing on the C50 and maximum expression values.
图1 图1

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


1) C.E. Outten, F.W. Outten, T.V. O’Halloran (1999) DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli, The Journal of Biological Chemistry, Vol. 274, No. 53, Issue of December 31, pp. 37517–37524.