Generator

Part:BBa_K1404002

Designed by: Alexandre Duprey   Group: iGEM14_INSA-LYON   (2014-09-23)

Ptac-NiCoTB, improves nickel and cobalt internalization

This part comprises a strong E. coli promoter with an heterologous metal transporter in order to give E. coli Ni2+ and Co2+ accumulation capacities in order to create a biofilter. This part works best in rcnA backgrounds (Ni2+ and Co2+ efflux defective)


Characterization

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

This part increases the internalization of Ni2+ and Co2+. Characterization was performed inside a more complex assembly comprising also an adherence operon ( K1404001)

A nikA E. coli MG1655 strain (S59) was compared to the same strain transformed with a plasmid containing K1404001 (pIG50). NikA is the main endogeneous nickel intake protein in E. coli, and it could have interfered with the measurement, which is the reason we inactivated it. Moreover, the experiment was performed in the presence of an excess of magnesium, to avoid nonspecific Ni2+ uptake by magnesium transporters.

The strains were cultured in LB medium to an OD600 of 0.6, at which time 150 nM 63NiCl2 was added. The cells were recovered after 0, 2.5, 5, 10, 15, 20 and 30 minutes of contact by filtration using a nitrocellulose filter. The filter was washed twice with EDTA in order to eliminate all extracellular nickel. The captured nickel was estimated by measuring the radioactivity of the filter. Data represent the mean of 2 replicates, and error bars represent standard deviations.

While the intracellular nickel remains nearly constant at 25 pmol Ni/mg BDW (Bacterial Dry Weight) in S59, the addition of the K1404002 allows the cells to accumulate up to 90 pmol Ni/mg BDW (6 µg/g BDW). Equilibrium seems to be attained after 30 mn. This result shows that this part is efficient at increasing the bacterial intracellular levels of Ni2+.

Further results concerning the overall efficiency of the biofilter 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 857
    Illegal AgeI site found at 1056
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//binding/metal
//cds/membrane/transporter
//chassis/prokaryote/ecoli
Parameters
None