Part:BBa_K1670000

CepI C8-HSL-sender device

This part encodes for the homoserine lactone-synthase of the CepR/I-system from Bulkholderia cepacia that produces octanoyl-homoserine lactone (C8-HSL). Upstream of the start codon a strong ribosome-binding site is already placed.

Characterization

BBa_K1670000 is cloned into J61002_BBa_J23100 for constitutive expression of the homoserine lactone synthases CepI (Fig.1). To verify functional expression of the synthase, single cell colonies are streaked on LB in parallel with Chromobacterium violaceum CV026. Native Chromobacterium violaceum produces the characteristic purple pigment violacein. In CV026 violacein production is inducible by multiple homoserine lactones with different sensitivities [1]. In consequence violacein is only produced, if BBa_K1670000 shows to be functional. As positive controls 0.5 μl of pure homoserine lactones in concentrations ranging from 10 mM to 1 nM were spotted next to C. violaceum. As negative controls wildtype BL21 and Nissle 1917 as well as 0.5 μl ddH₂O were used.

Fig. 1 . - J61002_BBa_J23100_BBa_K1670000 (CepI)

Results

In the positive controls (Fig. 2) one can see, that quite high homoserine lactone concentrations are needed to induce violacein expression. Octanoyl homoserine lactone induces reporter expression at concentrations of 100 µM and more.As expected in the negative controls no violacein expression can be observed in our reporter strain (Fig.3).

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Fig. 2 . - C. violaceum induced with different concentrations of octanoyl-homoserine lactone and grown over night at 30°C. A minimum of 100 µM C8-HSL is needed for violacein induction. C.v.: Chromobacterium violaceum CV026

Fig. 3 . - C. violaceumstreaked along E.coli BL21 E.coli Nissle 1917 as well as 0.5 µl ddH₂O spotted on the LB-plate and and grown over night at 30°C. Violacein cannot be observed due to a lack of homoserine lactones. C.v.: Chromobacterium violaceum CV026

BBa_K1670000 expression is putatively not functional in both used E.coli strains (Fig. 4) or the produced C8-HSL concentration is too low to induce violacein expression in C.violaceum as no violacein synthesis in C. violaceum can be observed.

Fig. 4 . - BBa_K16700040 is constitutively expressed in E.coli BL 21 and E.coli Nissle 1917. BBa_K1670000 cannot synthesize octanoyl-homoserine lactone in sufficient concentrations to induces violacein expression in C.violaceum . C.v.: Chromobacterium violaceum CV026

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 426
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1) McClean et al (1997) Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones; Microbiology 143 p.3703-3711