Part:BBa_K1520506

PgolTS-golS-PgolB

It is a gold ion detector. Gold ions will cause the CDS after PgolB to be expressed. PgolTS works like a constant promoter, producing gols which is inhibitor of Pgols. Once combining gold ions, gols will stop inhibitor PgolB. So, when there are gold ions, the CDS after this part will be expressed.

PgolTS-golS-PgolB works as a promoter, which will result in the expression of downstream gene in the presence of gold ions. As shown in (Fig. 1b), the gene of red fluorescence protein (RFP) was inserted after the PgolTS-golS-PgolB in the vector pSB1C3, allowing its expression to be driven by PgolTS-golS-PgolB, the gold-specific regulator.

The E. coli cells containing the engineering plasmid were cultured in LB medium until OD600=0.6–0.8, then a final concentration of 20 mM Au3+, Cu2+, Cd2+, Zn2+, or Ni2+ was added to the medium. The induced E. coli cells were harvested by centrifugation and re-suspended in PBS buffer (pH 7.4). The results showed that in the presence of gold ions, RFP was significantly expressed and can be visualized by the naked eye (Fig. 1c).

Next, the sensitivity of our engineered E. coli cells to gold was investigated. E. coli cells induced by gradient concentrations (0 mM, 0.25 mM, 1 mM, 5 mM, 10 mM and 20 mM) of HAuCl4 (Au3+) were re-suspended in PBS buffer (pH 7.4). The results demonstrated that even the induced concentration of gold ions reached as low as 0.25 mM (Fig. 1d, e), the red fluorescence of E. coli cells was still visible.

a) Genetic organization of the gol locus in the S. typhimurium LT2 genome. b) Genetic organization of the gold inductive RFP expression plasmid in E. coli. c) A photograph of E. coli cells containing the gold-induced RFP expression plasmid with a concentration of 20 mM each of metal ions, or without metal ions induction and re-suspended in PBS buffer (pH 7.4). d) Fluorescence measurement of E. coli cells containing the goldinduced RFP expression plasmid after gradient concentrations of HAuCl4 (Au3+) induction and re-suspended in PBS buffer (pH 7.4). e) A visible photograph of d.

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 548
  
• 1000
  COMPATIBLE WITH RFC[1000]