Part:BBa_K5323899

P_tac-RBS-yedQ-TT

Uses a strong promoter Ptac (BBa_K180000), a factor strong RBS(BBa_J34801), yedQ(BBa_K4242004) and TT(BBa_B0015). This part is an easy BioBrick.

Usage and Biology

This component is used to overexpress DGC in the engineering bacterium Acidithiobacillus ferrooxidans and participate in screening the most efficient cyclic diguanylate dynamic synthesis strains. We cloned the highly efficient DGC yedQ in E.coli and DGC AFE_0053, AFE_1373, AFE_1379 in A. ferrooxidans genome into the expression vector pYDT to construct the highly efficient expression vector of DGC. Furthermore, the carrier is conjured into A. ferrooxidans, the concentration of biofilm and c-di-GMP is measured, and the most efficient dynamic regulatory gene of c-di-GMP is screened.

Fig 1 Acidithiobacillus ferrooxidans pore plate biofilm formation column Fig 2 Escherichia coli pore plate biofilm formation column Fig 3 Schematic diagram of c-di-GMP content of E.coli BL21 YDT, AFE_1373, AFE_1379, YedQ. A is the c-di-GMP content of E.coli BL21 YDT, AFE_1373, AFE_1379, YedQ. Fig 4 Indicates the standard curve of c-di-GMP

Since it is previously known that IPTG can promote the significant expression of biofilm, after obtaining the growth characteristics of A. ferrooxidans, we directly measure the biofilm content in different time periods when the inducer IPTG is added to the strain. pYedQ was conducive to biofilm formation, and its significant difference P < 0.05 also indicated that its significantly promoted biofilm formation (Fig 3). In addition, we also determined the c-di-GMP content of E.coli BL21 transferred into plasmids pAFE_1373, pAFE_1379, pYedQ, and no-load pYDT. We calculated the c-di-GMP content according to the standard curve (Fig 4). With the addition of inducer IPTG, the c-di-GMP contents of pAFE_1373, pAFE_1379, pYedQ were significantly increased except for no-load plasmids.

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 AgeI site found at 3066
  
• 1000
  COMPATIBLE WITH RFC[1000]