Difference between revisions of "Part:BBa K3431007"

NUPACK analysis suggested the MFE (minimum free energy) RNA structure at 37℃, whose free energy was -22.00 kcal/mol. As for the Vienna binding, the black line in the figure indicates the amount of energy required to open the secondary structures of the TBS. The line red indicates the amount of energy required to open the secondary structure after the binding of the trigger. As a result, this indicates that zr31 will successfully be bound and open the locked structure.

Characterization using invertase

The 2020 iGEM CSMU-Taiwan characterized the zr31 toehold switch with T7 promoter (BBa_I719005), invertase reporter protein (BBa_K3431000), and T7 terminator(BBa_K731721). We built up a composite part BBa_K3431023 to test its functionality. 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”.

Fig. 3. The glucose productions of the zr31 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-31 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-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, the zr31 toehold switch has been proven to be useful for miR-31 detection.

Characterization using invertase

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.

Fig. 4. Glucose production under different amounts of miR-31.
Results
As shown above, the glucose concentration rose as the miR-31 triggers increased, representing a positive correlation.

References

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Sequence and Features