Part:BBa_K1104202

HemH promoter

Introduction

HemHp promoter is controlled by OxyR (transcription factor), which is activated by ROS(Reactive Oxygen Species). An intramolecular disulfide bond between Cys199 and Cys208 will form in an OxyR transcription factor, and active OxyR molecule will bind to the binding site of HemHp, making the following circuit turned on.

In our project: Bee. coli, we use HemH as the sensor of Bee. coli to sense Nosema ceranae. After Nosema ceranae enter bees' midgut, the natural immune reaction will lead to ROS released by epithelia of bees' midgut. We select several promoters that can be induced by OxyR, which is activated by ROS. One of these promoters is HemHp.

HemHp

What is 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. 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.}

Information of HemHp on [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104 Ecocyc]

We get information of HemHp from [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104 Ecocyc],

HemHp is regulated by σ70, and there are two TFBSs upstream for OxyR. We also did some literature study, according to the citation.

In E. coli genome, thioredoxin(trxC) will express in response to elevated H₂O₂ level. Expression of genes encoding thioredoxin(trxC) is regulated by OxyR. Oxidized OxyR binds directly to HemHp and induce its expression.

Schematic drawing of the HemH regulon in E. coli K12 (taken from [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104 Ecocyc])

Nucleotide Sequence of HemHp(taken from [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104 Ecocyc])

OxyR TFBS1 Sequence of HemHp(taken from [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104 Ecocyc])

OxyR TFBS2 Sequence of HemHp(taken from [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104 Ecocyc])

_{Reference: Ritz, D. (January 28, 2000). Thioredoxin 2 Is Involved in the Oxidative Stress Response in Escherichia coli. Journal of Biological Chemistry, 275, 4, 2505-2512.}

Usage in Our Project

We designed circuit fighting against Nosema ceranae. After Nosema ceranae infected midgut cells of bees, and Bee. coli should sense the pathogen first before the following circuit(fighting against Nosema ceranae)is triggered, and substance such as Defensin(Part:BBa_K1104300), Abaesin(Part:BBa_K1104301) (more details on [http://2013.igem.org/Team:NYMU-Taipei/Project/Inhibition/Killing Killing Nosema] page) in the following circuit will express.

Besides, in order to enhance the strength of HemHp, we added a device (more details on [http://2013.igem.org/Team:NYMU-Taipei/Project/Inhibition/Sensor Sensing Nosema] page).

HemHp-strenthening device

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]