Part:BBa_K2012000

c-di-GMP tandem riboswitche bc3-5

Three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5′-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43.

Illustration of riboswitch mechanism

As show in the illustration, transcript of riboswitches region would form a hairpin and terminate transcription under low concentration of c-di-GMP.

Schematic of riboswitches

(a)Comparison of Bc3, Bc4, Bc5 terminators. Red rectangle shows the GC rich region of three terminators, respectively. (b) Secondary structure comparisons of Bc3, Bc4 and Bc5 aptamers with Vc2 aptamer. Conserved motifs such as tetra-loop (blue motif in stem P2), tetra-loop receptor (green motif in stem P3) and G·C base pair (C base in stem P2 and G base in stem P3 were drawn in magenta) connecting P2 with P3 were all colored to facilitate comparison. c-di-GMP was drawn in cyan and its interacting bases drawn in red.(Zhou et al., 2016) (c) Multiply local sequence blast of riboswitches’ terminators. Blue rectangle shows U region of riboswitches.

Congo red staining

For demonstrating expression of pleD, we used Congo red staining assay. As previous mentioned, high concentration of c-di-GMP could induce E. coli synthesize exopolysaccharides, and Congo red binding is a complex phenotype that reflects various outer membrane and surface properties including the presence of adhesive structures such as curli fimbria which are involved in biofilm formation. Wild type: DE3 contain pET28b plasmid, colony which was stained red color contain pET-pleD plasmid.

Reference:

Zhou, H., Zheng, C., Su, J., Chen, B., Fu, Y., Xie, Y., . . . He, J. (2016). Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter. Sci Rep, 6, 20871. doi:10.1038/srep20871

Usage and Biology

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]