Part:BBa_K1670004

EsaI 3OC6-HSL sender device

BBa_K1670004 (EsaI) encodes for the homoserine lactone – synthase of the EsaR/I system from Erwinia stewartii that produces 3-oxo-hexanoyl-homoserine lactone (3OC6-HSL). Upstream of the start codon a ribosome-binding site is already placed.

Characterization

BBa_K1670004 is cloned into J61002_BBa_J23100 for constitutive expression of the homoserine lactone synthases EsaI (Fig.1). To verify functional expression of the synthases, single cell colonies are streaked on LB-plates 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_K1670004 shows to be functional. As a positive control 3-oxohexanoyl-homoserine lactone dissolved in acetonitrile and diluted with ddH₂O to a concentration of 100 nm is spotted next to C. violaceum CV026 respectively. As a negative control ddH₂O is spotted.

Fig. 1 . - J61002_BBa_J23100_BBa_K1670004 (EsaI)

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).

Fig. 2 . - C. violaceum induced with different concentrations of 3-oxohexanoyl-homoserine lactone and grown over night at 30°C. A minimum of 1 mM 3OC6-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_K1670004 seems to be functional in both E.coli strains (Fig.4). As expected the expression strain E.coli BL21 shows a better result, due to knocked out protease activity. Still E.coli Nissle 1917 produces enough BBa_K1670004 to induce weak violacein expression in C. violaceum . This confirm functionality of BBa_K1670004.

Fig. 4 . - BBa_K1670004 is constitutively expressed in E.coli BL 21 and E.coli Nissle 1917. BBa_K1670004 synthesizes 3-oxohexanoyl homoserine lactone that 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
  COMPATIBLE WITH RFC[25]
• 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