Difference between revisions of "Part:BBa K2116011"

Latest revision as of 17:37, 28 October 2016

AND gate regulated by Nitric Oxide and 3OC6HSL

The NorV promoter (PnorV, Part:BBa_K1153000) is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. coli [1]. Its transcriptional regulator, NorR Part:BBa_K256004, can sense nitric oxide and activate gene expression.

EsaR Part:BBa_K2116001 is a transcriptional regulator of the P. stewartii quorum sensing system [2]. 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, kanamycine resistance). We employ 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 constructed AND gate shows favorable behaviour (Figure 1), reaching a 4-fold increase in sfGFP expression after induction with DETA/NO and AHL.

Figure 1: 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.

Figure 2: 3D plot representing AND gate behaviour. Different AHL and NO concentrations were tested and GFP fluorescence was measured. Black dots represent experimental data. The surface represents interpolation between the experimental data points. Highest fluorescence intensity is reached at highest concentrations of AHL and NO, while lower concentrations result in lower fluorescence intensity.

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) (Figure 3) [3]. 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.

Figure 3: A norR KO strain was used as a negative control to demonstrate that the native norR of E.coli is enough to sufficiently 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] Minogue, Timothy D., et al. "The autoregulatory role of EsaR, a quorum‐sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function." Molecular microbiology 44.6 (2002): 1625-1635.
• [3] 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