Part:BBa_K3386001

MphR is a repressor protein of Mph(A)

Usage and Biology

MphR is a repressor protein binding pMphR. It downregulates the transcription of 2′-phosphotransferase Mph(A). It contains the TetR-type HTH DNA-binding domain [1]. Upon binding of two erythromycin molecules it undergoes a conformational change and unbinds from pMphR, allowing for Mph(A) transcription [2].

MphR can be used in systems requiring regulation of pMphR.

Characterization

We have designed a system where MphR was driven by a constitutive promoter. pMphR was driving the expression of egfp (Fig. 1). We transformed Escherichia coli TOP10 strain with this system and grew the cells for 20 h in LB with various erythromycin concentrations. We have also grown cells transformed with a construct lacking mphr gene, to see how much fluorescence can be produced in absence of the repressor. We observed repression of egfp transcription, since the amount of fluorescence produced by cells not expressing the repressor was visibly the highest. There also seems to be a correlation between the amount of erythromycin present and the pMphR promoter activation, which suggests MphR is functional and unbounds the DNA upon ligand binding (Fig. 2).

Figure 1. Genetic circuit designed to assess the functionality of pMphR.

Figure 2. Optical density (OD) normalized fluorescence values in different erythromycin (ERY) concentrations (0, 0.1, 0.5, 1, 10, 50, 100 µg/ml). The graph includes data from three repeats.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 492
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 386
  Illegal XhoI site found at 37
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]