Difference between revisions of "Part:BBa K3431003"
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| − | === | + | ===Characrterization using invertase=== |
| + | The 2020 iGEM CSMU-Taiwan characterized the toehold switch with invertase (BBa_K3431000) reporter protein. The plasmid would be transcribed and translated with the protein synthesis kit at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose | ||
| + | concentration with Rightest TM GS550 glucose meter after 30 minutes. In our experiments, the ON state refers to the conditions with miRNA triggers; while | ||
| + | the OFF state means that there was no miRNA in the environment. We calculated the ON/OFF ratio of the toehold switch, which is defined as “the glucose concentration of the ON state/ the glucose concentration of the OFF state”. | ||
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<img src="https://static.igem.org/mediawiki/parts/d/d1/T--CSMU_Taiwan--zp21_B_%28BBa_K3431018%29.png" style="width:50%"> | <img src="https://static.igem.org/mediawiki/parts/d/d1/T--CSMU_Taiwan--zp21_B_%28BBa_K3431018%29.png" style="width:50%"> | ||
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| + | Figure 3. The glucose productions of the zp21_B toehold switch-regulated invertase in different states. The blue bar refers to the OFF state (not added with miRNA). The green bar refers to the ON state (added with miR-21 trigger). The yellow bar refers to the state with non-related RNAs (added with miR-191). The pink bar refers to the state with non-related RNAs (added with miR-223). | ||
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| + | <b>Results</b> The ON/OFF ratio with miR-21 is 0.99, which suggested the leakage problem. The regulatory function of the zp21_B toehold switch was not good | ||
| + | enough. Thus, zp21_B toehold switch-regulated invertase cannot be well controlled by the miR-21. | ||
===References=== | ===References=== | ||
Revision as of 11:01, 25 October 2020
zp21_B Toehold Switch for miR-21 Detection
Introduction
zp21_B toehold switch is a regulatory part for the downstream reporter gene. With this part, the protein expression can be controlled by the miR-21. The sequence of the toehold switch can be separated into the following 5 regions from its 5' end: TBS (trigger binding site), stem region, loop region with RBS(ribosome binding site), complimentary stem region with a start codon, and linker. Upon binding with miR-21, its hairpin structure can be opened up and the ribosomes can bind with its RBS (ribosome binding site), triggering the translation of the downstream reporter.
Design
The design of the toehold switch was mainly based on the previous research. 1 2 3 4 5 6 For the zp21_B toehold switch, we adopted the loop structure from Green et al., 2016 7, and the linker structure is from Wang et al., 2019 8 . Using NUPACK analysis and Vienna binding models, we designed the sequence of the toehold switch. (See our model page:https://2020.igem.org/Team:CSMU_Taiwan/Model)
Characrterization using invertase
The 2020 iGEM CSMU-Taiwan characterized the toehold switch with invertase (BBa_K3431000) reporter protein. The plasmid would be transcribed and translated with the protein synthesis kit at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose
concentration with Rightest TM GS550 glucose meter after 30 minutes. In our experiments, the ON state refers to the conditions with miRNA triggers; while
the OFF state means that there was no miRNA in the environment. We calculated the ON/OFF ratio of the toehold switch, which is defined as “the glucose concentration of the ON state/ the glucose concentration of the OFF state”.
Results The ON/OFF ratio with miR-21 is 0.99, which suggested the leakage problem. The regulatory function of the zp21_B toehold switch was not good enough. Thus, zp21_B toehold switch-regulated invertase cannot be well controlled by the miR-21.
References
Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: de-novo-designed regulators of gene expression. Cell, 159(4), 925-939. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., ... & Daringer, N. M. (2016). Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell, 165(5), 1255-1266. Wang, S., Emery, N. J., & Liu, A. P. (2019). A novel synthetic toehold switch for microRNA detection in mammalian cells. ACS synthetic biology, 8(5), 1079-1088.
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]