Part:BBa_K1031931

J23114-XylC-Terminator

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Characterization

BBa_K1031931 is composed of three elements, the constitutive Pc promoter J23114, coding sequence of XylC and terminator B0015. Two rounds of site mutation have been finished to remove an EcoR1 site and a Xba1 site to meet RFC10. (Fig 1)

Fig 1 Construction of adaptor circuit J23114-XylC. The orange arrowhead refers to constitutive Pc promoter. RBS(Ribosome Binding Site) is shown in green oval. The square in dark red stands for terminator B0015.

Sequence and Features

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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1012
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1349
  
• 1000
  COMPATIBLE WITH RFC[1000]

Data shown

The induction effect of 3-methyl-benzaldehyde and 3-chlorobenzaldehyde on XylS combined with adaptor XylC improved significantly comparing to biosensor XylS only (Fig. 2a). Furthermore, the dose-response curves for 3-methyl-benzaldehyde of Biosensor XylS and XylS equipped with XylC (Fig. 2b) showed that the Adaptor XylC could reduce the detection limit (the concentration of inducer at which an output three times the basal single is generated) of 3-methyl-benzaldehyde from 100 μM to 1 μM. Similarly, the detection limit of 3-chloro-benzaldehyde was decreased more than 1 order of magnitude (Fig. 2c). In addition, dose-response curves for 3-methyl-benzoate and 3-chlorobenzoate were also collected (Fig. 2d). The almost identical lines for 3-methyl-benzoate and 3-chloro-benzoate showed that adding Adaptor XylC does not significantly influence the original characteristics of the biosensor.

Fig 2 (a) Fluorescence intensity of XylS and XylS equipped with Adaptor XylC elicited by 3-methyl-benzaldehyde and 3-chloro-benzaldehyde at the concentration of 1 mM. XylS with Adaptor XylC showed higher fluorescence intensity and induction ratio than XylS. (b) Dose-response curves of XylS and XylS equipped with XylC for 3-methyl-benzaldehyde. Use of Adaptor XylC significantly reduced the detection limit by 100 folds. (c) Dose-response curves of XylS and XylS equipped with XylC for 3-chloro-benzaldehyde. (d) Dose-response curves of XylS and XylS equipped with XylC for 3-methyl-benzoate and 3-chloro-benzoate. The almost overlapping curves for the two compounds showed that the Adaptor XylC did not interfere the performance of XylS biosensor.