Difference between revisions of "Part:BBa K1153000"

Revision as of 23:55, 16 October 2016

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 Parts:BBa_K2116002, and includes a spacer that is not found in this version.

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) 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.

Sequence and Features