Difference between revisions of "Part:BBa K2973014"
Akatsaouni (Talk | contribs) |
|||
| (4 intermediate revisions by 3 users not shown) | |||
| Line 2: | Line 2: | ||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K2973014 short</partinfo> | <partinfo>BBa_K2973014 short</partinfo> | ||
| − | 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 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>Dictyoglomus turgidum</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. | ||
This trigger sequence was derived from the 16S rRNA sequence of <i>Dictyoglomus turgidum</i>. | This trigger sequence was derived from the 16S rRNA sequence of <i>Dictyoglomus turgidum</i>. | ||
| − | |||
===Usage and Biology=== | ===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 ,<i>in silico</i>. One of them was Toehold No.14. The reporter gene attached to the toehold switch is β-lactamase. | + | 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.14. The reporter gene attached to the toehold switch is β-lactamase. |
| − | 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. The major issue with toehold switches is their leakiness (translation in absence of trigger sequence), so we tested only one trigger concentration. | + | 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 at 37 ℃. The major issue with toehold switches is their leakiness (translation in absence of trigger sequence), so we tested only one trigger concentration. |
As seen in Figure 1, the signal produced by Toehold 14 is very similar to that of the positive control. However, when no trigger sequence is added, the signal produced is the same, if not higher, than the 100nM trigger condition. This can be interpreted as a lack of regulation of translation by the riboswitch. That means that the toehold switch has an open conformation and thus an accessible RBS, even when no trigger is added. | As seen in Figure 1, the signal produced by Toehold 14 is very similar to that of the positive control. However, when no trigger sequence is added, the signal produced is the same, if not higher, than the 100nM trigger condition. This can be interpreted as a lack of regulation of translation by the riboswitch. That means that the toehold switch has an open conformation and thus an accessible RBS, even when no trigger is added. | ||
Latest revision as of 17:37, 21 October 2019
Trigger for Toehold switch 14 Dictyoglomus turgidum
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 Dictyoglomus turgidum.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 Dictyoglomus turgidum.
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.14. 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 at 37 ℃. The major issue with toehold switches is their leakiness (translation in absence of trigger sequence), so we tested only one trigger concentration.
As seen in Figure 1, the signal produced by Toehold 14 is very similar to that of the positive control. However, when no trigger sequence is added, the signal produced is the same, if not higher, than the 100nM trigger condition. This can be interpreted as a lack of regulation of translation by the riboswitch. That means that the toehold switch has an open conformation and thus an accessible RBS, even when no trigger is added.
Figure 1: Bars show the ability of toehold 14 to hydrolyze nitrocefin. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 64
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]