Difference between revisions of "Part:BBa K1104200"

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OxyR

Introduction

OxyR is Escherichia coli transcription factor that senses H₂O₂ to activate transcription, and is activated through the formation of an intramolecular disulfide bond. The OxyR gene, a positive regulatory gene for the oxidative response, then controls proteins which are induced by treatment with H₂O₂.

OxyR (BBa_K1104200)

Literature Study of OxyR

Illustration of OxyR Activation

OxyR, a positive regulator of hydrogen peroxide-inducible genes in E. coli. The OxyR transcription factor regulates the H₂O₂ response of E. coli.

Oxidative stress, through the production of Reactive Oxygen Species (ROS) , is a natural consequence of aerobic metabolism. Escherichia coli has several major regulators activated during oxidative stress, including OxyR, SoxRS, and RpoS. OxyR undergo conformation changes when oxidized in the presence of hydrogen peroxide, then positively controls the expression of cognate genes.

_{Reference: Chiang, S. M., Schellhorn, H. E., & Catalases and Hydrogen Peroxide Metabolism. (September 15, 2012) . Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Archives of Biochemistry and Biophysics, 525, 2, 161-169.}

H₂O₂ oxidizes OxyR Cys199 to an R-SOH that reacts with Cys208 in an intramolecular disulphide bond.

Structure of the OxyR Regulatory Domain
(A) Reduced Form: Inactive
(B) Oxidized Form: Activated by ROS

_{Reference: D'Autréaux, B., & Toledano, M. B. (October 01, 2007). ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nature Reviews Molecular Cell Biology, 8, 10, 813-824.}

Usage and Biology

After Nosema ceranae enter bees' midgut, the natural immune reaction will lead to ROS released by epithelia of bees' midgut. In our project: Bee. coli, we use OxyR-induced promoter as the sensor of Bee. coli to sense Nosema ceranae. We select several promoters that can be induced by OxyR(Part:BBa_K1104200).

There are promoters that can be activated by OxyR: TrxCp(Part:BBa_K1104201), HemHp (Part:BBa_K1104202) , ahpC (Part:BBa_K362001) , sufA (Part:BBa_K362005) , and dps (Part:BBa_K362002) , yiaA (Part:BBa_K362006) .

The OxyR-induced promoters:

• TrxCp (Part:BBa_K1104201) and HemHp (Part:BBa_K1104202) are parts created by our team [http://2013.igem.org/Team:NYMU-Taipei/Modeling/MainParts NYMU_Taipei]. (Come to the [http://2013.igem.org/Team:NYMU-Taipei/Experiment/Wet_Lab Wet Lab] page to see our part list.)

• sufA (Part:BBa_K362005) , designed by [http://2010.igem.org/Team:KIT-Kyoto/Parts 2010 KIT-Tokyo team], is successfully characterized again by us.

• ahpC (Part:BBa_K362001) is improved its complex composition and Assembly Compatibility by us. We designed ahpC (Part:BBa_K362001) improvement into 6 versions:

In order to enhance the strength and sensitivity of ROS-sensing promoters, we make E. coli continuously produce inactivated OxyR transcription factors, so that ROS can affect promoters more easily.

We designed a device that is composed of a sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp1000, AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp) plus reporter (Part:BBa_E0840). Device1 in order to enhance the effect of ROS on E. coli is added ahead: a constitutive promoter(Part:BBa_J23102), and OxyR(Part:BBa_K1104200).

Contribution: Improvement and Characterization

Team SUIS_Shanghai 2019 attempted to improve the sensitivity of this OxyR to reactive oxygen species (ROS) by increasing the accessible surface area of the reactive cysteine (cysteine-199) within the protein. This cysteine, once oxidized will form a intermolecular disulfide bond with Cys-208, resulting in a conformational change which can activate certain ROS sensitive promoters and therefore positively expressing genes in times of oxidative stress.

Related Parts

Sequence and Features