Difference between revisions of "Part:BBa K4207065"
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Sensor plasmid for detecting BYDV | Sensor plasmid for detecting BYDV | ||
| − | + | ===1. Usage and biology=== | |
| − | ===Usage and | + | This sensor plasmid was designed to detect a conserved RNA sequence in the barley yellow dwarf virus (BYDV) genome. It should express the reporter protein β-galactosidase only in the presence of its specific trigger sequence. The translation is controlled by a toehold switch, which sequesters the RBS and the start codon in a stable RNA secondary structure. This structure is unfolded in the presence of the specific trigger, which is a conserved sequence in the BYDV genome. |
| + | |||
| + | The activity of the translated β-galactosidase can be measured spectrophotometrically if the reaction contains a colorimetric substrate for β-galactosidase. The construct can be used in various reaction set ups. If used in a PURE (protein expression using recombinant elements) system or any other environment with reduced nuclease activity, it can be used as a linear plasmid. However, if used in a reaction with active nucleases, for instance a lysate-based cell-free expression system, the construct should be used as a circular plasmid to reduce degradation. | ||
| + | |||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K4207065 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4207065 SequenceAndFeatures</partinfo> | ||
| + | |||
| + | ===2. Characterization=== | ||
| + | |||
| + | This part was tested for the production of β-galactosidase in E. coli Gold-dlac (DE3) lysate-based cell-free expression system (sCFE). We tested its functionality in two reactions in the absence and presence of its specific ssDNA. The plasmid backbone in these reactions was pOdd-1 (BBa_J428381).This sensor plasmid produced 0.56-fold activity of β-galactosidase in the presence of the trigger and so did exhibit slight trigger-dependency in its translational activity. The results are shown in Fig 1. | ||
| + | |||
| + | [[File:aboa-65.png|center|500px|Fig 1.]] | ||
| + | <b>Fig 1.</b> Toehold switch performance in sCFE system. sCFE reactions containing toehold sensor plasmids with β-galactosidase as the reporter protein were monitored by measuring the absorbance at 420 nm. Cell-free reactions containing the toehold sensor plasmid were measured in the presence or absence of its specific trigger ssDNA. The graph shows the absorbance over time in the reaction. Each data point represents the average of two parallel reactions and error bars represent standard deviation. | ||
| + | |||
| + | This toehold sensor plasmid exhibited 0.56-fold activity of β-galactosidase in the presence of its specific ssDNA-trigger. This sensor plasmid did not function as desired. | ||
| + | |||
| + | ===3. Conclusion=== | ||
| + | The experimental data about this sensor’s functionality is lacking. However, the combination of experimental and the modeling data about this sensor plasmid’s toehold switch suggests that this sensor plasmid is not likely to function as desired. | ||
Latest revision as of 15:35, 10 October 2022
Toehold sensor plasmid B46-LacZ
Sensor plasmid for detecting BYDV
1. Usage and biology
This sensor plasmid was designed to detect a conserved RNA sequence in the barley yellow dwarf virus (BYDV) genome. It should express the reporter protein β-galactosidase only in the presence of its specific trigger sequence. The translation is controlled by a toehold switch, which sequesters the RBS and the start codon in a stable RNA secondary structure. This structure is unfolded in the presence of the specific trigger, which is a conserved sequence in the BYDV genome.
The activity of the translated β-galactosidase can be measured spectrophotometrically if the reaction contains a colorimetric substrate for β-galactosidase. The construct can be used in various reaction set ups. If used in a PURE (protein expression using recombinant elements) system or any other environment with reduced nuclease activity, it can be used as a linear plasmid. However, if used in a reaction with active nucleases, for instance a lysate-based cell-free expression system, the construct should be used as a circular plasmid to reduce degradation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
2. Characterization
This part was tested for the production of β-galactosidase in E. coli Gold-dlac (DE3) lysate-based cell-free expression system (sCFE). We tested its functionality in two reactions in the absence and presence of its specific ssDNA. The plasmid backbone in these reactions was pOdd-1 (BBa_J428381).This sensor plasmid produced 0.56-fold activity of β-galactosidase in the presence of the trigger and so did exhibit slight trigger-dependency in its translational activity. The results are shown in Fig 1.
Fig 1. Toehold switch performance in sCFE system. sCFE reactions containing toehold sensor plasmids with β-galactosidase as the reporter protein were monitored by measuring the absorbance at 420 nm. Cell-free reactions containing the toehold sensor plasmid were measured in the presence or absence of its specific trigger ssDNA. The graph shows the absorbance over time in the reaction. Each data point represents the average of two parallel reactions and error bars represent standard deviation.
This toehold sensor plasmid exhibited 0.56-fold activity of β-galactosidase in the presence of its specific ssDNA-trigger. This sensor plasmid did not function as desired.
3. Conclusion
The experimental data about this sensor’s functionality is lacking. However, the combination of experimental and the modeling data about this sensor plasmid’s toehold switch suggests that this sensor plasmid is not likely to function as desired.