Part:BBa_K2116002

Nitric oxide responsive norV promoter

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

We cloned PnorV upstream of super folder GFP for characterisation. This construct was expressed on a medium copy plasmid (p15A). Since E.coli natively produce NorR, we relied on this to activate the promoter. Below is the dose response curve we obtained.

NorV promoter activated by nitric oxide, released from DETA/NO. Above 15000uM of DETA/NO affects cell growth and is not included in the dose response.

In order to ascertain that we are using the native NorR and not another transcriptional regulator, we used a Keio norR knock-out strain (norR KO) [2] 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 of E.coli can activate PnorV in the AND gate.

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. [2] Baba, Tomoya, et al. "Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection." Molecular systems biology 2.1 (2006).

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]