Part:BBa_K2940014

XylR coding device-sfGFP_optimism coding device with Pu

This part encodes the biosensor genetic circuit comprising the toluene-specific transcription factor XylR and the sfGFP reporter gene.
This part is a modification of the BBa_K2023015 part originally developed by Ionis Paris iGEM team 2016.
The part was meant to serve as the starting biosensor circuit platform for the subsequent directed evolution of XylR in order to develop a biosensor for the detection of aromatic amines.

Usage and Biology

BTEX (Benzene, Toluene, Ethylbenzene, Xylene)-like aromatic compounds detection.

Characterization

The E. coli TOP10 cells transformed with XylR:sfGFP_pSB1C3 biosensor plasmid exhibited strong fluorescence even when uninduced. This strong background fluorescence was most presumably the result of sfGFP leaky expression and made cells apparently not sensitive to the induction when grown on plain and toluene-containing plates (Figure 1).

Figure 1. Screening for toluene-sensitive cells on LB plates without or with toluene inducer. Toluene was used at the 10 mg/L final concentration.

Indeed, there were no significant differences in fluorescence responses between uninduced and toluene-induced cells when accurately measured by a plate reader (Figure 2A). The situation was not improved even by inverting the direction of the XylR sensing module and the sfGFP reporter module, so they face opposite directions and their transcription is really independent (Figure 2B), nor when using the low-copy pSB3C5 and pSB4C5 plasmid backbones (Figure 2C).

Figure 2. Biosensor variants inducibility by toluene. (A) The original XylR:sfGFP_pSB1C3 biosensor variants. (B) XylR_inv:sfGFP_pSB1C3 biosensor variants with opposite module orientation. (C) Low-copy XylR:sfGFP_pSB3C5/pSB4C5 biosensor variants.

Conclusion

Our results suggest, that the codon-optimized sfGFP variant might not be a suitable reporter in certain biosensor settings, as any leaky expression can create a very strong fluorescence background signal obscuring the actual signal.

Sequence and Features

Assembly Compatibility:

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

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2101
  Illegal XhoI site found at 3018
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 329
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Unknown