Part:BBa_K3431016
pp21_B_ToeholdSwitch-Regulated Invertase
Introduction
pp21_B_ToeholdSwitch-Regulated Invertase is a genetic device that can be applied as a biosensor for miRNA. It is designed to detect and measure the amount of miR-21 by the expression of Thermotoga maritima Invertase (BBa_K3431000). The invertase can convert sucrose to glucose, which can be easily measured by a personal glucose meter (PGM).
Components
pp21_B_ToeholdSwitch-Regulated Invertase consists of 4 basic parts: T7 promoter (BBa_I719005), pp21_B toehold switch (BBa_K3431001), invertase (BBa_K3431000), and T7 terminator (BBa_K731721). The mechanism of the detection is mainly based on the regulatory part, pp21_B Toehold Switch for miR-21 Detection (BBa_K3431001). 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 process of the downstream reporter, invertase (BBa_K3431000). As for the T7 promoter (BBa_I719005) and T7 terminator (BBa_K731721), they are the essential genetic elements for the PURExpress protein synthesis kit.
Construction
The 2020 iGEM CSMU-Taiwan used in-fusion cloning to construct the composite part. The process is shown below.
Response in different miRNA
To further understand its functionality, 2020 iGEM CSMU-Taiwan conducted a series of tests. 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 RightestTM 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 0.96, which suggested the leakage problem. The regulatory function of the pp21_B toehold switch was not good enough. Thus, pp21_B toehold switch-regulated invertase cannot be well controlled by the miR-21.
Reference
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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1425
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1196
Illegal BamHI site found at 1326
Illegal XhoI site found at 1397 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 997
- 1000COMPATIBLE WITH RFC[1000]
None |