Part:BBa_K5323894

P_tac-RBS-golS-P_gol-RBS-gfp-TT

Uses a strong promoter P_tac (BBa_K180000), two factor strong RBS(BBa_J34801), golS (BBa_K4468005), P_gol(BBa_K4468001) ,gfp(BBa_E0040) and TT(BBa_B0015). This part is an easy BioBrick.

Usage and Biology

This component is used to test the specificity and sensitivity of gold binding.We used gfp as the target gene to verify the specificity and sensitivity of Au[Ⅲ] by detecting the fluorescence intensity emitted by bacteria in cultures with different metals and different Au[Ⅲ] concentrations. We examined the growth characteristics and fluorescence content of E.coli BL21 strain in different ions with the same concentration (Fig. 1A, B). It can be observed that the response of strains to Au[Ⅲ] is very significant. The average fluorescence intensity of the strains with Au[Ⅲ] ions in the middle and late growth was significantly higher than that of other ions (Fig 1B). In addition, we also tested the sensitivity of gold binding. The control group was set up with the addition of inducer 0.5mM IPTG and different concentrations of HAuCl4, and the same experiment was performed with different concentrations of HAuCl4 and without the addition of inducer IPTG. The results showed that E.coli BL21 strain, which transformed pYDT-golS-P_gol-gfp, could respond to lower gold ion concentration (Fig 2B). With the increase of Au[Ⅲ] concentration, E.coli BL21 showed a more significant response to Au[Ⅲ] (Fig 2B).

Fig. 1 Growth characteristics and expression characteristics of E.coli BL21 in response to Au. A is the growth curve of E.coli BL21 in different metal ions with the same concentration. B is a diagram of the average fluorescence intensity of E.coli BL21 in different metal ions with the same concentration. C is the average fluorescence intensity diagram of E.coli BL21 cultured in different metal ions with the same concentration for 12 hours. Fig. 2 Growth characteristics and expression characteristics of E.coli BL21 sensitivity response to Au. A is the growth curve of E.coli BL21 in different concentrations of Au[Ⅲ]. B is the schematic diagram of the average fluorescence intensity of E.coli BL21 in different concentrations of Au[Ⅲ]. C is the average fluorescence intensity diagram of E.coli BL21 cultured in different concentrations of Au[Ⅲ] for 12 hours.

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 1635
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3721
  Illegal AgeI site found at 1969
  Illegal AgeI site found at 2318
  Illegal AgeI site found at 3256
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4570