Part:BBa_K1404001

Ptac-NiCoTB-rcn-csgBAEFG, turns E. coli into a functional Ni and Co biofilter

This part, when expressed in a rcnA E. coli strain, allows the formation of a functional Ni²⁺ and Co²⁺ biofilter capturing up to 3 mg of metal per g of bacterial dry weight when exposed to 20 µM Ni²⁺ or Co²⁺, in only 10 mn. This is one of the best performances and the fastest reported until now.

Characterization

This part and the relevant data were published in [1]. A summary of the most significant findings is presented below. Note that pIG50 is K1404001 in pSB1T3.

Adherence

This part gives the cells a constitutive adherence that can be further enhanced by the presence of nickel or cobalt.

A constitutively Gfp producing strain (S29) and its engineered version S61 (S29 rcnA/K1404001) were compared in their adherence capacities by fluorescence microscopy (A) or confocal microscopy (B)

The strains were incubated in 96-well plates for 24h at 30°C in minimal medium supplemented with various metal concentrations as indicated on the figure.

In fluorescence microscopy (A), in the absence of metal, S61 displayed a mild aggregative behaviour compared to the wild-type strain. In the presence of metal, the formation of biofilms by S61 was enhanced, while no change was observed for S29. In confocal microscopy (B), the thickness of the biofilms was estimated. The engineered strains were significantly thicker in the presence of metal (n=5, p<0,005, Dunnett's test), which quantitatively confirmed our observations with fluorescence microscopy.

Metal uptake

This part improves the metal capture of E. coli, however most metal captured by the filter is bound nonspecifically by the bacterial extracellular matrix.

The S29 (WT) and S61 S61 (S29 rcnA/K1404001) strains were cultured for 24 hours in minimal medium in Petri dishes. The medium was then removed and replaced by a metal solution prepared in sterile water. After 10 mn (A) or 30 mn (B/C) of contact between the biofilm and the metal solution, the supernatant was discarded. The captured metal was measured by ICP-MS (Inductively Coupled Plasma Mass Spectrometry).

Absolute metal capture yields without washing the biofilm with EDTA (A) show that the strain harbouring K1404001 (S61) performs overall slightly better than a wild-type strain, especially in the range 0-20 nM for nickel and 12-50 nM for cobalt.

Further characterization was performed at a fixed concentration of 10 µM metal. Washing the biofilm with EDTA (B and C) revealed that most captured metal was in fact extracellular. B shows the relative contribution of specific internalization of metal, defined as the ratio of metal recovered from EDTA-washed cells (dashed lines) to unwashed cells (plain colors). If considering only the specific uptake of nickel and cobalt, i.e. by washing the biofilm with EDTA (C), K1404001 allows a significantly higher metal capture, which could be useful in the presence of much more complex effluents containing high concentrations of metal ions like Fe²⁺, which would compete with Ni²⁺ and Co²⁺ for nonspecific binding to the extracellular matrix.

Further results are available in the associated article

References

[1] Duprey A, Chansavang V, Fremion F, Gonthier C, Louis Y, Lejeune P, Springer F, Desjardin V, Rodrigue A, Dorel C: “NiCo Buster”: engineering E. coli for fast and efficient capture of cobalt and nickel. J Biol Eng 2014, 8:19.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1361
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 857
  Illegal AgeI site found at 1056
  Illegal AgeI site found at 3422
  
• 1000
  COMPATIBLE WITH RFC[1000]