Difference between revisions of "Part:BBa K4207002"
 
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<partinfo>BBa_K4207002 short</partinfo>
 
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Toehold switch for the detection of BYDV gRNA
 
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===1. Usage and Biology===
 
===1. Usage and Biology===
  
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Toehold switches are de novo designed riboregulators that can be used to sense different nucleic acid sequences. They are specifically designed RNA sequences that have the ribosome binding site (RBS) and the start codon in a stem-loop followed by a reporter gene. The RBS and the start codon are sequestered in the secondary structure, which hinder the translation of the reporter gene. The toehold switch has a specific binding site to its trigger sequence, which extends to the base of the stem-loop. When the trigger binds, it unwinds the lower part of the stem-loop, leaving only a weak secondary structure intact. This remaining structure is designed to be weak, so ribosome binding unwinds the structure, allowing translation to occur. (Green et. al., 2014) (Green et. al., 2017).
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Toehold switches are engineered riboregulators that control the expression of a downstream protein coding sequence. They can be designed to detect virtually any sequence. Toehold switches are designed <i>in silico</i> so that they fold into a pre-determined secondary structure. This structure contains a stable stem-loop that sequesters the ribosome binding site (RBS) and the start codon, thus preventing translation. After a specific trigger RNA binds to the binding site of the toehold, the lower part of the stem-loop unfolds, revealing the start codon. A weak stem remains, but this structure unfolds upon ribosome binding to the RBS, starting translation (Green et al., 2017). This toehold switch was designed to detect conserved sequences in the X genome. The structural change of the toehold switch is illustrated in Figure 1.
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Here we designed a A-series toehold switch, which has a structure allowing lower translational leakage to previous toehold switches (Pardee et. al., 2016). This toehold switch is designed to detect the presence of barley yellow dwarf virus gRNA by binding to a conserved sequence found in the virus' enome.
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<b>Figure 1</b>. Toehold switch mechanism. This animation illustrates the operation of the toehold switch. Initially, the structure is in an inactive state and the RBS and the start codon are hidden in the stem-loop. When a specific trigger binds to the binding site, the stem-loop structure opens and the ribosome binding site and start codon are revealed.
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To use this toehold switch, it should be assembled in a construct containing a promoter, the toehold switch, a protein-coding sequence, and optionally a terminator if the sensor is not to be used as linear. To prevent frame-shifting, the last nucleotide is omitted from the sequence and this part is compatible with iGEM Type IIS standard assembly.
  
 
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===2. Design===
 
===2. Design===
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This toehold switch was designed according to the A-series ideal structure from Pardee et al. (2016). This structure was improved from the original toehold switch structure (Green et al., 2014) to reduce translational leakage. We screened the BYDV genome for conserved sequences. Each sequence was divided into 36-nucleotide long subsequences and we designed toehold switches designed to specifically bind to the sequence. This toehold switch was designed using the 30-nucleotide linker found in the 27B sensor (Pardee et al., 2016). We assigned a score for each toehold switch based on the three-parameter fit from Ma et al. (2018) and selected the best-ranking toehold switches for our library.
  
 
===3. Characterization===
 
===3. Characterization===
  
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<b>Score predicted by our model: 16.41</b><br />
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This part was tested in part BBa_K4207061 for the production of β-galactosidase in E. coli Gold-dlac (DE3) lysate-based cell-free expression system. We tested its functionality in two reactions in the absence and presence of its specific ssDNA. This toehold switch produced 0.56-fold activity of β-galactosidase in the presence of the trigger and so did not exhibit the desired trigger-dependent translation.
  
 
===4. Conclusion===
 
===4. Conclusion===
  
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===5. References===
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The experimental data about this part is limited and the composite part was likely not optimized, so further experiments would be necessary to judge this part’s performance. However, based on the modeling and experimental data combined, we suggest that this toehold switch is not likely to function as desired. For this reason, we’d suggest using BBa_K4207012 or one of BBa_K4207014-BBa_K4207017, as they are more likely to detect the BYDV genome.
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 14:41, 10 October 2022


BYDV toehold switch A70


1. Usage and Biology

Toehold switches are engineered riboregulators that control the expression of a downstream protein coding sequence. They can be designed to detect virtually any sequence. Toehold switches are designed in silico so that they fold into a pre-determined secondary structure. This structure contains a stable stem-loop that sequesters the ribosome binding site (RBS) and the start codon, thus preventing translation. After a specific trigger RNA binds to the binding site of the toehold, the lower part of the stem-loop unfolds, revealing the start codon. A weak stem remains, but this structure unfolds upon ribosome binding to the RBS, starting translation (Green et al., 2017). This toehold switch was designed to detect conserved sequences in the X genome. The structural change of the toehold switch is illustrated in Figure 1.

Figure 1. Toehold switch mechanism. This animation illustrates the operation of the toehold switch. Initially, the structure is in an inactive state and the RBS and the start codon are hidden in the stem-loop. When a specific trigger binds to the binding site, the stem-loop structure opens and the ribosome binding site and start codon are revealed.

To use this toehold switch, it should be assembled in a construct containing a promoter, the toehold switch, a protein-coding sequence, and optionally a terminator if the sensor is not to be used as linear. To prevent frame-shifting, the last nucleotide is omitted from the sequence and this part is compatible with iGEM Type IIS standard assembly.

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]

2. Design

This toehold switch was designed according to the A-series ideal structure from Pardee et al. (2016). This structure was improved from the original toehold switch structure (Green et al., 2014) to reduce translational leakage. We screened the BYDV genome for conserved sequences. Each sequence was divided into 36-nucleotide long subsequences and we designed toehold switches designed to specifically bind to the sequence. This toehold switch was designed using the 30-nucleotide linker found in the 27B sensor (Pardee et al., 2016). We assigned a score for each toehold switch based on the three-parameter fit from Ma et al. (2018) and selected the best-ranking toehold switches for our library.

3. Characterization

Score predicted by our model: 16.41
This part was tested in part BBa_K4207061 for the production of β-galactosidase in E. coli Gold-dlac (DE3) lysate-based cell-free expression system. We tested its functionality in two reactions in the absence and presence of its specific ssDNA. This toehold switch produced 0.56-fold activity of β-galactosidase in the presence of the trigger and so did not exhibit the desired trigger-dependent translation.

4. Conclusion

The experimental data about this part is limited and the composite part was likely not optimized, so further experiments would be necessary to judge this part’s performance. However, based on the modeling and experimental data combined, we suggest that this toehold switch is not likely to function as desired. For this reason, we’d suggest using BBa_K4207012 or one of BBa_K4207014-BBa_K4207017, as they are more likely to detect the BYDV genome.