Part:BBa_K2116011

PnorV_esabox_(15bp)spacer_esabox_spacer_esabox (p30)

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

EsaR is a transcriptional regulator of the P. stewartii quorum sensing system. Unlike other quorum sensing regulators, it acts as a repressor and not an activator. It binds DNA through an 18bp binding site called esabox. When bound to 3OC6HSL, it is released to allow transcription.

We created an AND gate by placing one esabox as roadblock, and one esabox right before the first norR binding site of the PnorV promoter. This second esabox was added to create competitive binding between norR and esaR.

We have characterised the AND gate using constitutively expressed esaR on a medium copy plasmid (pRBR322/rop). We rely on the native NorR production in E. coli to activate the AND gate. The AND gate controls the expression of sfgfp Part:BBa_K2116017, and is on a medium copy plasmid (p15A).

The AND gate was tested in presence of DETA/NO and 3OC6HSL. Activation was quantified through GFP expression. The AND gate works as expected, reaching maximal expression only when both NO and AHL are present at the highest concentration tested.

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) and compared it to its parent wild type strain (WT) [2]. In this case only PnorV was tested, which can also be found on the registry Part:BBa_K2116002. 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]