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<p>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&#8242;-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43.</p>
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<p>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&#8242;-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43.
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They are c-di-GMP riboswitches (termed c-di-GMP-I), c-di-GMP-I
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binds the second messenger c-di-GMP through hydrogen bonding or base stacking of the two guanosines of
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c-di-GMP with five specific bases in the aptamer, resulting in transcriptional changes of downstream genes correlating
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with c-di-GMP function.</p>
 
<img src="https://static.igem.org/mediawiki/parts/4/49/Illustration_of_riboswitch_mechanism.png" width="600px"/>
 
<img src="https://static.igem.org/mediawiki/parts/4/49/Illustration_of_riboswitch_mechanism.png" width="600px"/>
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<h1>Illustration of riboswitch mechanism</h1>
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            </br> <b> Figure 1.</b> Illustration of riboswitch mechanism. As shown in the illustration, transcript of riboswitches region would form a hairpin and terminate transcription under low concentration of c-di-GMP.
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<p>As show in the illustration, transcript of riboswitches region would form a hairpin and terminate transcription under low concentration of c-di-GMP. </p>
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<img src="https://static.igem.org/mediawiki/parts/6/66/Schematic_of_riboswitches.png"  width="800px"/>
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<img src="https://static.igem.org/mediawiki/parts/d/d5/Bc4.5.png"  width="450px"/>
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<h1>Schematic of riboswitches</h1>
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<p>(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.</p>
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<p><b>Figure 2.</b> Schematic of riboswitches. (a) Comparison of Bc3, Bc4, Bc5 terminators. Red rectangle shows the GC rich region of three terminators, respectively. (b) Multiply local sequence blast of riboswitches’ terminators. Blue rectangle shows U region of riboswitches.</p>
 
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<img src="https://static.igem.org/mediawiki/parts/3/3d/Congo_red_stain.png"  width="600px"/>
 
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<h1>Congo red staining</h1>
 
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<p>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.</p>
 
 
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<img src="https://static.igem.org/mediawiki/parts/5/50/Characterization2.png"  width="800px"/>
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<p><b>Figure 3.</b> Characterization of Riboswitch. PC: pET-28-pleD\J23106+sfGFP  64F: pET-28-pleD\ J23106+Bc3+sfGFP 65F: pET-28-pleD\ J23106+Bc5+sfGFP  644F: pET-28-pleD\ J23106+Bc4+Bc4+sfGFP  635F: pET-28-pleD\ J23106+Bc3-5+sfGFP  74F: pET-28-pleD\ J23117+Bc3+sfGFP
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75F: pET-28-pleD\ J23117+Bc5sfGFP  735F: pET-28-pleD\ J23117+Bc3-5+sfGFP.
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<h1>Reference:</h1>
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<p>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</p>
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<img src="https://static.igem.org/mediawiki/parts/b/bb/Characterization3.png"  width="600px"/>
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<p><b>Figure 4.</b> Characterization of Riboswitch. We use “read-through rate” (RTR) of the downstream gene to measure the terminator forming efficiency of a riboswitch. The read-through rate of each riboswitch in liquid medium was assessed by relative fluorescence intensity, which is the ratio of specific activity of a test strain to specific activity of the control strain (pET-28-pleD/J23117+sfGFP) with the same promoter of test circuits (such as J23117+Bc3-5+sfGFP).
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Latest revision as of 13:38, 1 November 2016


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. They are c-di-GMP riboswitches (termed c-di-GMP-I), c-di-GMP-I binds the second messenger c-di-GMP through hydrogen bonding or base stacking of the two guanosines of c-di-GMP with five specific bases in the aptamer, resulting in transcriptional changes of downstream genes correlating with c-di-GMP function.


Figure 1. Illustration of riboswitch mechanism. As shown in the illustration, transcript of riboswitches region would form a hairpin and terminate transcription under low concentration of c-di-GMP.



Figure 2. Schematic of riboswitches. (a) Comparison of Bc3, Bc4, Bc5 terminators. Red rectangle shows the GC rich region of three terminators, respectively. (b) Multiply local sequence blast of riboswitches’ terminators. Blue rectangle shows U region of riboswitches.




Figure 3. Characterization of Riboswitch. PC: pET-28-pleD\J23106+sfGFP 64F: pET-28-pleD\ J23106+Bc3+sfGFP 65F: pET-28-pleD\ J23106+Bc5+sfGFP 644F: pET-28-pleD\ J23106+Bc4+Bc4+sfGFP 635F: pET-28-pleD\ J23106+Bc3-5+sfGFP 74F: pET-28-pleD\ J23117+Bc3+sfGFP 75F: pET-28-pleD\ J23117+Bc5sfGFP 735F: pET-28-pleD\ J23117+Bc3-5+sfGFP.




Figure 4. Characterization of Riboswitch. We use “read-through rate” (RTR) of the downstream gene to measure the terminator forming efficiency of a riboswitch. The read-through rate of each riboswitch in liquid medium was assessed by relative fluorescence intensity, which is the ratio of specific activity of a test strain to specific activity of the control strain (pET-28-pleD/J23117+sfGFP) with the same promoter of test circuits (such as J23117+Bc3-5+sfGFP).


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]