Difference between revisions of "Part:BBa K3431035"

Characterization 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 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”.

Figure. 3. The glucose productions of the zr146_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-146 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).

Results
The glucose concentration in the ON state with miR-146 is about 500 mg/dL, indicating the high sensitivity of the toehold switch. The ON/OFF ratio with miR-146 is 1.21, which suggested the regulatory function of the toehold switch. By contrast, the ON/OFF ratios with miR-191 and miR-223 are 0.82 and 1.00, respectively. These ratios are close to 1, meaning the zr146_B toehold switch has high specificity. As a result, zr146_B_ToeholdSwitch-Regulated Invertase has been proven to be useful for miR-146 detection.

References

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