Difference between revisions of "Part:BBa K5106004"
Ivargruppen (Talk | contribs) (Add qualitative test results and general info) |
|||
| Line 3: | Line 3: | ||
<partinfo>BBa_K5106004 short</partinfo> | <partinfo>BBa_K5106004 short</partinfo> | ||
| − | MS toehold A (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106001 BBa_K5106001]</span> under control of a T7 promoter and terminator, regulating expression of a LacZ reporter gene, in order to test the toehold functionality ''in vitro'' in a cell-free PURExpress system | + | MS toehold A (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106001 BBa_K5106001]</span> under control of a T7 promoter and terminator, regulating expression of a LacZ reporter gene, in order to test the toehold functionality ''in vitro'' in a cell-free PURExpress system. |
| − | + | ==Usage and Biology== | |
| − | === | + | This composite part consists of the basic toehold switch part (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106001 BBa_K5106001]</span>), under control of a <span class="plainlinks">[https://parts.igem.org/Part:BBa_K1614000 T7 promoter]</span> and <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106019 terminator]</span>, and the <span class="plainlinks">[https://parts.igem.org/Part:BBa_K1444017 LacZ reporter gene]</span>. To test the whether the designed toehold switch works, a plasmid expressing this composite part was made, and expressed in a PURExpress cell-free system. This is a reconstituted form of cell-free expression that works by isolating the necessary components, such as ribosomes and amino acids, purifying them, and assembling them into a reaction mixture.<sup>'''1'''</sup> |
| + | |||
| + | ===Qualitative ''in vitro'' test=== | ||
| + | To first check whether the toehold switch was activated by the trigger miRNA hsa-miR-484 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106000 BBa_K5106000]</span>), we performed a test in 2 μL PURExpress, with ~25 ng plasmid containing the toehold switch (this composite part). Besides other toehold switches during this test, a negative control, containing no DNA template, and a positive control, containing only ''lacZ'' under the control of the T7 promoter and terminator, were included as well. In addition, 0.5 μL miRNA <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106000 hsa-miR-484]</span> (5 μM) was added to part of the samples. The tubes were incubated at 37 °C for two hours. | ||
| + | |||
| + | <html> | ||
| + | <br> | ||
| + | <div style=”width:100%; display:block; align-items: center; justify-content: center;"> | ||
| + | <img src="https://static.igem.wiki/teams/5106/parts-figures/tha-tube.png" style="width:60%; align=center> | ||
| + | </div> | ||
| + | <br> | ||
| + | </html> | ||
| + | <small>'''Figure 1:'''Cell-free expression (PURExpress) of toehold switch constructs in 2 μL reaction volumes. To the samples in the top row, no trigger miRNA was added. To the samples in the bottom row, 0.5 μL miRNA was added. From left to right: negative control; toehold switch 1 (not used); toehold switch 2(not used); MS-specific toehold switch A; MS-specific toehold switch B; MS-specific toehold switch C; toehold switch AND gate; positive control. A red box is place around the samples of MS toehold switch A, discussed on this part. For more details of this experiment, take a look on the website of <span class="plainlinks">[https://2024.igem.wiki/wageningenur/wet-lab-results miRADAR (WageningenUR 2024)]</span>. </small> | ||
| + | |||
| + | After two ours, β-galactosidase (LacZ) performed as expected, since no colour change from yellow to purple was observed in the negative control, whereas the colour change was observed in the positive control (Figure 1). This change is caused by For the part containing MS toehold switch A, a clear colour change can also be observed (Figure 1), indicating that the toehold switch can indeed be activated by hsa-miR-484, resulting in further translation of the downstream protein coding region. | ||
<!-- --> | <!-- --> | ||
| − | <span class='h3bb'>Sequence and Features</span> | + | =<span class='h3bb'>Sequence and Features</span>= |
<partinfo>BBa_K5106004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5106004 SequenceAndFeatures</partinfo> | ||
| Line 17: | Line 31: | ||
<partinfo>BBa_K5106004 parameters</partinfo> | <partinfo>BBa_K5106004 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| + | |||
| + | |||
| + | 1. Shimizu, Yoshihiro, Akio Inoue, Yukihide Tomari, Tsutomu Suzuki, Takashi Yokogawa, Kazuya Nishikawa, and Takuya Ueda. ‘Cell-Free Translation Reconstituted with Purified Components’. Nature Biotechnology 19, no. 8 (August 2001): 751–55. https://doi.org/10.1038/90802. | ||
Revision as of 12:42, 1 October 2024
Toehold switch A for detection of hsa-miR-484 with T7 promoter and terminator, and LacZ reporter
MS toehold A (BBa_K5106001 under control of a T7 promoter and terminator, regulating expression of a LacZ reporter gene, in order to test the toehold functionality in vitro in a cell-free PURExpress system.
Usage and Biology
This composite part consists of the basic toehold switch part (BBa_K5106001), under control of a T7 promoter and terminator, and the LacZ reporter gene. To test the whether the designed toehold switch works, a plasmid expressing this composite part was made, and expressed in a PURExpress cell-free system. This is a reconstituted form of cell-free expression that works by isolating the necessary components, such as ribosomes and amino acids, purifying them, and assembling them into a reaction mixture.1
Qualitative in vitro test
To first check whether the toehold switch was activated by the trigger miRNA hsa-miR-484 (BBa_K5106000), we performed a test in 2 μL PURExpress, with ~25 ng plasmid containing the toehold switch (this composite part). Besides other toehold switches during this test, a negative control, containing no DNA template, and a positive control, containing only lacZ under the control of the T7 promoter and terminator, were included as well. In addition, 0.5 μL miRNA hsa-miR-484 (5 μM) was added to part of the samples. The tubes were incubated at 37 °C for two hours.
miRADAR (WageningenUR 2024).
After two ours, β-galactosidase (LacZ) performed as expected, since no colour change from yellow to purple was observed in the negative control, whereas the colour change was observed in the positive control (Figure 1). This change is caused by For the part containing MS toehold switch A, a clear colour change can also be observed (Figure 1), indicating that the toehold switch can indeed be activated by hsa-miR-484, resulting in further translation of the downstream protein coding region.
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]
1. Shimizu, Yoshihiro, Akio Inoue, Yukihide Tomari, Tsutomu Suzuki, Takashi Yokogawa, Kazuya Nishikawa, and Takuya Ueda. ‘Cell-Free Translation Reconstituted with Purified Components’. Nature Biotechnology 19, no. 8 (August 2001): 751–55. https://doi.org/10.1038/90802.