Regulatory

Part:BBa_K1153000

Designed by: Kara Stubbs, David Hanly, Laura Carman, Sarah Dowie, Michael Coghlan, Rathaven Gunaratnarajah.   Group: iGEM13_Kent   (2013-09-20)
Revision as of 14:45, 22 October 2016 by Aasli (Talk | contribs)

NorV promoter. NOx detector.

Our Biobrick has been designed to enable the detection of NO using the norV gene promoter, cloned from E.coli K12 TOP10 cells. In the presence of NOx it will promote the downstream genes in the plasmid, producing a protein in response to the environmental stimulus.

Contribution

  • Group: ETH Zurich 2016
  • Author: Asli Azizoglu
  • Summary: We cloned and characterised the norV promoter, and sent it to the registry as a biobrick. Our biobrick can be found here Part:BBa_K2116002, and includes a spacer that is not found in this version.

Usage and Biology

Promoter norV (PnorV) is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. Coli. [1] It's transcriptional regulator, NorR, can bind to nitric oxide and activate gene expression.


Characterisation of the Promoter

We cloned PnorV upstream of superfolder GFP for characterisation Part:BBa_K2116088 This construct was expressed on a medium copy plasmid (ori:p15A). Since E.coli natively produces NorR, we relied on this to activate the promoter. Below is the dose response curve we obtained under the following experimental conditions, using a plate reader:

  • Overnight growth and experiment in minimal M9 medium, with 25μg/μL chloramphenicol.
  • Plating at an OD600 of 0.05, in M9, with 25μg/μL chloramphenicol.
  • Induction at OD6000.5.
  • Settings for GFP measurement: excitation-488nm, emission- 530nm.
  • Samples were always in three technical replicates, and the fluorescence measurements were normalized to OD600.
PnorV dose response curve for a range of DETA/NO concentrations that corresponds to 7-70μM of NO. Above 15000uM of DETA/NO affects cell growth and is not included in the dose response. Measured 6 hours after induction. Error bars represent S.D. from three technical replicates.

In order to show that the native NorR is essential for activating PnorV, we used a <a href=http://cgsc.biology.yale.edu/KeioList.php>Keio</a> norR knock out strain (ΔnorR) and compared it to it's parent wild type strain (WT). It was shown that PnorV can be activated by DETA/NO in the parent strain, but not in the norR KO strain.

A norR KO strain was used as a negative control to demonstrate that the native norR can activate PnorV.Induced with 5000μM DETA/NO and measured 6 hours after induction. Error bars represent S.D. from three technical replicates.

References:

[1] Gardner, A. M. "Regulation Of The Nitric Oxide Reduction Operon (Norrvw) In Escherichia Coli. ROLE OF Norr AND Sigma 54 IN THE NITRIC OXIDE STRESS RESPONSE". Journal of Biological Chemistry 278.12 (2003): 10081-10086.


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]


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Categories
Parameters
None