Part:BBa_K3431023
zr31_ToeholdSwitch-Regulated Invertase
Description
zr31_ToeholdSwitch-Regulated Invertase is a genetic device that can be applied as a biosensor for miR-31, which is a biomarker of oral squamous cell carcinoma (OSCC)[1][2][3][4].This part consists of 4 basic parts: T7 promoter (BBa_I719005), zr31 Toehold Switch (BBa_K3431007), Thermotoga maritima Invertase (BBa_K3431000), and T7 terminator (BBa_K731721).
The mechanism of the detection is mainly based on the regulatory part, zr31 Toehold Switch for miR-31 Detection (BBa_K3431007). When the miR-31 binds with the trigger binding site of the toehold, the hairpin structure can be opened up and the ribosomes can bind with the RBS, triggering the translation of the downstream reporter, invertase (BBa_K3431000). The invertase can convert sucrose to glucose, which can be easily measured by a personal glucose meter (PGM).
As for the T7 promoter (BBa_I719005) and T7 terminator (BBa_K731721), they are the essential genetic elements for the in vitro gene expression with PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs).
Construction
The construction process of the composite part is shown below.
Response in different miRNA
To further understand the functionality of the genetic device, 2020 iGEM CSMU-Taiwan conducted a series of tests. The plasmids were transcribed and translated with the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs) at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose, and measured the glucose concentration with Bionime Rightest™ GM550 glucose meter after 30 minutes of enzymatic reaction time. 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 glucose concentration in the ON state with miR-31 is 310.67 mg/dL, indicating the high sensitivity of the toehold switch. The ON/OFF ratio with miR-31 is 2.65, which suggested the regulatory function of the toehold switch. By contrast, in the experiment of negative selection, the ON/OFF ratios with miR-191 and miR-223 are 1.46 and 1.21, respectively. These ratios are close to 1, meaning the zr31 toehold switch has high specificity. As a result, zr31_ToeholdSwitch-Regulated Invertase has been proven to be useful for miR-31 detection.
Response under different amounts of trigger
To understand the correlation of the trigger amount and the glucose production, we added different amounts of miR-31 to the protein synthesis kit and produced the proteins at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose concentration with the glucose meter after 30 minutes.
Results
As shown above, the glucose concentration rose as the miR-31 triggers increased, representing a positive correlation.
Reference
1. Liu, C.-J., Lin, S.-C., Yang, C.-C., Cheng, H.-W., & Chang, K.-W. (2011). Exploiting salivary miR-31 as a clinical biomarker of oral squamous cell carcinoma. Head & Neck, 34(2), 219–224. https://doi.org/10.1002/hed.21713
2. Mazumder, S., Datta, S., Ray, J. G., Chaudhuri, K., & Chatterjee, R. (2019). Liquid biopsy: miRNA as a potential biomarker in oral cancer. Cancer epidemiology, 58, 137–145. https://doi.org/10.1016/j.canep.2018.12.008
3. Min, A., Zhu, C., Peng, S., Rajthala, S., Costea, D. E., & Sapkota, D. (2015). MicroRNAs as Important Players and Biomarkers in Oral Carcinogenesis. BioMed Research International, 2015, 1–10. https://doi.org/10.1155/2015/186904
4. Momen-Heravi, F., Trachtenberg, A. J., Kuo, W. P., & Cheng, Y. S. (2014). Genomewide Study of Salivary MicroRNAs for Detection of Oral Cancer. Journal of Dental Research, 93(7_suppl), 86S-93S. https://doi.org/10.1177/0022034514531018
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1426
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1197
Illegal BamHI site found at 1327
Illegal XhoI site found at 1398 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 998
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 529
None |