Part:BBa_K3386004

Biosensor device for erythromycin detection

Usage and Biology

The genetic circuit comprises a BBa_J23106 constitutive promoter (BBa_J23106) driving expression of mphr (BBa_K3386001) and ermC (BBa_K3386000), as well as egfp (BBa_K3386003) under pMphR promoter (BBa_K3386002). MphR and ermC are preceded by a BBa_B0029 ribosomal binding site. The egfp gene is preceeded by the BBa_K3386005 ribosomal binding site. The terminator used was BBa_B0029 (Fig.1).

MphR is a repressor protein containing TetR-type HTH DNA-binding motif [1]. It binds to pMphR - a promoter region driving the expression of 2′-phosphotransferase Mph(A). Upon binding of two erythromycin molecules MphR releases the DNA and allows transcription of genes dependent on pMphR promoter [2]. ErmC is methyltransferase, which introduces dimethylation of the adenine residue in 23S rRNA at position 2085, thus preventing binding of macrolide and lincosamide antibiotics [3].

The device can be used for erythromycin detection. In presence of erythromycin cells expressing this composite part will produce a fluorescent signal which can be correlated to the amount of compound present in the sample.

Characterization

To assess functionality of the biosensor we performed a plate-reader experiment, where we grew cells transformed with the biosensor device in various erythromycin concentrations for 20 h. Non-transformed E. coli grown in LB with 100 µl erythromycin were a positive control. A negative control comprised non-transformed cells grown in LB with no antibiotic. Optical density and fluorescence were measured every 15 minutes. The GFP intensity was normalized using OD600 measurements. We performed three repeats. We have observed a correlation between concentration of erythromycin in the sample and intensity of fluorescent signal. However the differences between these signals are not very significant (Fig. 2).

Figure 1. Schematic overview of the genetic circuit.

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 7
  Illegal NheI site found at 30
  Illegal NheI site found at 542
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 436
  Illegal XhoI site found at 87
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2353