Regulatory

Part:BBa_K3431039

Designed by: Jian-An Pan, Cheng-Yang Ma, Yi-Ching Chen, Shen-Lin Chen, Huan-Jui Chang   Group: iGEM20_CSMU_Taiwan   (2020-10-17)
Revision as of 14:38, 26 October 2020 by Ivychen (Talk | contribs) (→‎Model)


Banana Toehold Switch

Introduction

The sequence of this part is from a modular synthetic biology education kit,banana toehold sensor sfGFP (Biobits) 1 . It is a regulatory part that can be triggered by the RNA of the banana and express the downstream reporter.

Model

We used NUPACK analysis to see its spontaneous formation of the secondary structure at 37℃, and used Vienna Binding to predict the interaction between the toehold with the trigger.

Figure 1. NUPACK analysis result
Figure. 2. ViennaRNA Package result

















Characterization using sfGFP

To test the functionality of the banana toehold switch, 2020 iGEM CSMU_Taiwan used sfGFP (BBa_K3431031) as the reporter protein. We measured the real-time fluorescence of the sfGFP within the 80 minutes protein production process. The result is shown below.

Figure 1. The relative fluorescence of the sfGFP in the 80 minutes protein production process. The relative fluorescence was measured by the Synergy H1 Hybrid Multi-Mode Reader. The green line refers to the conditions with banana triggers, while the blue line refers to the conditions without a banana trigger.

Results As seen, the sfGFPs would be highly expressed when the banana triggers existed, while they would be inhibited when there was no trigger in the environment. This suggested the regulatory function of the toehold switch.

Characterization using invertase

The 2020 iGEM CSMU_Taiwan also used Thermotoga maritima Invertase (BBa_K3431000) as the reporter to test the functionality of the banana toehold switch. 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 the Rightest TM GS550 glucose meter every 10 minutes at 55℃ for an hour. In our experiment, the ON state refers to the conditions with miRNA triggers; while the OFF state means that there was no miRNA in the environment.

Figure 2. The glucose concentration in 60 minutes invertase enzymatic reaction. The green line refers to the conditions with banana triggers, while the blue line refers to the conditions without a banana trigger.

Results As shown above, glucose concentration increased with time in the ON state but remained low in the OFF state. The result suggested the regulatory function of the banana toehold switch.

Reference

1. Huang, A., Nguyen, P. Q., Stark, J. C., Takahashi, M. K., Donghia, N.,Ferrante, T., Dy, A. J., Hsu, K. J., Dubner, R. S., Pardee, K., Jewett, M. C., & Collins, J. J. (2018). BioBits™ Explorer: A modular synthetic biology education kit. Science advances, 4(8), eaat5105.https://doi.org/10.1126/sciadv.aat5105

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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