Difference between revisions of "Part:BBa K2116011"

Revision as of 20:36, 16 October 2016

AND gate regulated by Nitric Oxide and 3OC6HSL

Promoter norV 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.

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 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.

proof that native norR works.

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]

@@ Line 9: / Line 9: @@
 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 (pRB322/rop). We rely on the native NorR production in E.coli to activate the AND gate.
+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.
 [[File:Bba K2116011 AND gatebehaviour.png|500px|thumb|center|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.]]