Difference between revisions of "Part:BBa K2973013"
 
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<partinfo>BBa_K2973013 short</partinfo>
 
<partinfo>BBa_K2973013 short</partinfo>
  
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Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. Upstream of the coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 5’ end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single stranded region (36bp) that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the coding gene.
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This composite part consists of T7 Promoter (<partinfo>BBa_J64997</partinfo>), the T7 Terminator (<partinfo>BBa_K731721</partinfo>) and the trigger sequence derived from the 16S rRNA sequence of <i>Geobacillus kaustophilus</i>. Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. Upstream of the coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 5’ end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single-stranded region (36bp) that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the coding gene.
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This trigger sequence was derived from the 16S rRNA sequence of Geobacillus kaustophilus.  
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This trigger sequence was derived from the 16S rRNA sequence of <i>Geobacillus kaustophilus</i>.  
  
  
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A key aspect of our project is to provide a tool that can detect the DNA of any organism easily and reliably. To that end, we designed, a pool of universal toehold switches from hyperthermophile bacteria ,<i>in silico</i>. One of them was Toehold No.13. The reporter gene attached to the toehold switch is β-lactamase.
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A key aspect of our project is to provide a tool that can detect the DNA of any organism easily and reliably. To that end, we designed, a pool of universal toehold switches from hyperthermophile bacteria, <i>in silico</i>. One of them was Toehold No.13. The reporter gene attached to the toehold switch is β-lactamase.
  
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To assess the new toehold's performance, we performed a series of <i>in vitro</i> protein synthesis reactions. The in vitro transcription/ translation reactions were done using the PURExpress® <i>In Vitro</i> Protein Synthesis kit. To reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL. The incubation time was 3 hours.  
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To assess the new toehold's performance, we performed a series of <i>in vitro</i> protein synthesis reactions. The <i>in vitro</i> transcription/ translation reactions were done using the PURExpress® <i>In Vitro</i> Protein Synthesis kit. To reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL. The incubation time was 3 hours. After the 3-hour incubation in the cell-free system, the chromogenic substrate of β-lactamase, nitrocefin, was added and an additional 15-minute enzymatic assay was performed in the plate reader, at 37&#8451;. 
  
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The performance of our designed Toehold 13, compared to the 32B Toehold ([[Part:BBa_K2973007]]), is depicted below. We have also added a non-regulated lactamase CDS as a positive control
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The performance of our designed Toehold 13 is depicted below.
  
 
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       <img src="https://static.igem.org/mediawiki/parts/d/da/Toehold_13.png" width="800"
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       <img src="https://2019.igem.org/wiki/images/2/2a/T--Thessaly--Registry_Toehold13.png" width="800"
 
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<b>Figure 1.</b> Performance of toehold 13 in a β-lactamase enzymatic assay. Error bars represent standard deviation of n = 2 replicates.
 
<b>Figure 1.</b> Performance of toehold 13 in a β-lactamase enzymatic assay. Error bars represent standard deviation of n = 2 replicates.
  
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The results were successful because Toehold 13 was able to regulate the translation of β-lactamase in absence of trigger, while the 100nM trigger reaction reached easily the positive control signal.  
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The expression of β-lactamase under the regulation of Toehold 13 was noticeably low in the absence of a trigger sequence, while the 100nM trigger reaction easily reached levels of signal comparable to the positive control.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 17:35, 21 October 2019


Trigger for Toehold switch 13 Geobacillus kaustophilus

This composite part consists of T7 Promoter (BBa_J64997), the T7 Terminator (BBa_K731721) and the trigger sequence derived from the 16S rRNA sequence of Geobacillus kaustophilus. Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. Upstream of the coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 5’ end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single-stranded region (36bp) that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the coding gene. This trigger sequence was derived from the 16S rRNA sequence of Geobacillus kaustophilus.


Usage and Biology

A key aspect of our project is to provide a tool that can detect the DNA of any organism easily and reliably. To that end, we designed, a pool of universal toehold switches from hyperthermophile bacteria, in silico. One of them was Toehold No.13. The reporter gene attached to the toehold switch is β-lactamase.

To assess the new toehold's performance, we performed a series of in vitro protein synthesis reactions. The in vitro transcription/ translation reactions were done using the PURExpress® In Vitro Protein Synthesis kit. To reduce the cost of the reaction, we lowered the reaction volume from 25 to 7 μL. The incubation time was 3 hours. After the 3-hour incubation in the cell-free system, the chromogenic substrate of β-lactamase, nitrocefin, was added and an additional 15-minute enzymatic assay was performed in the plate reader, at 37℃.

The performance of our designed Toehold 13 is depicted below.

HTML img Tag

Figure 1. Performance of toehold 13 in a β-lactamase enzymatic assay. Error bars represent standard deviation of n = 2 replicates.

The expression of β-lactamase under the regulation of Toehold 13 was noticeably low in the absence of a trigger sequence, while the 100nM trigger reaction easily reached levels of signal comparable to the positive control.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 64
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]