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===Construction===
 
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The 2020 iGEM CSMU-Taiwan used in-fusion cloning to construct the composite part. The process is shown below.
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The construction process of the composite part is shown below.
 
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Figure. 1. In-fusion cloning of the toehold switch regulated invertase. (A) Using PCR to produce the target insert, which includes invertase and T7 terminator sequences. The forward primer contained XbaI and overlapped with the 5’ end of the invertase; while the reverse primer contained PstI and was complementary to the 3’ end of the T7 terminator. (B) Lane 1 to 7 are the toehold switch vectors digested with XbaI and PstI, whose length is about 2000 bp. Lane 11 is the Insert containing invertase and T7 terminator, whose length is 1358 bp. (C) Using in-fusion cloning technology to ligate the invertase with the toehold switches we designed.
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Figure. 1. Gene cloning of the toehold switch regulated invertase. (A) Using PCR to produce the target insert, which includes invertase and T7 terminator sequences. The forward primer contained XbaI and overlapped with the 5’ end of the invertase; while the reverse primer contained PstI and was complementary to the 3’ end of the T7 terminator. (B) Lane 1 to 8 are the toehold switch vectors digested with XbaI and PstI, whose length is about 2000 bp. Lane 9 is the Insert containing invertase and T7 terminator, whose length is 1358 bp. (C) Ligate the invertase sequence with the toehold switches we designed.
 
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<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
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Latest revision as of 06:32, 27 October 2020


zr146_A_ToeholdSwitch-Regulated Invertase

Introduction

zr146_A_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-146 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

zr146_A_ToeholdSwitch-Regulated Invertase consists of 4 basic parts: T7 promoter (BBa_I719005), zr146_A toehold switch (BBa_K3431011), invertase (BBa_K3431000), and T7 terminator (BBa_K731721). The mechanism of the detection is mainly based on the regulatory part, zr146_A Toehold Switch for miR-146 Detection (BBa_K3431011). Upon binding with miR-146, 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 construction process of the composite part is shown below.

Figure. 1. Gene cloning of the toehold switch regulated invertase. (A) Using PCR to produce the target insert, which includes invertase and T7 terminator sequences. The forward primer contained XbaI and overlapped with the 5’ end of the invertase; while the reverse primer contained PstI and was complementary to the 3’ end of the T7 terminator. (B) Lane 1 to 8 are the toehold switch vectors digested with XbaI and PstI, whose length is about 2000 bp. Lane 9 is the Insert containing invertase and T7 terminator, whose length is 1358 bp. (C) Ligate the invertase sequence with the toehold switches we designed.

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”.

Figure. 2. The glucose productions of the zr146_A_ToeholdSwitch-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 450 mg/dL, indicating the high sensitivity of the toehold switch. The ON/OFF ratio with miR-146 is 2.07, which suggested the regulatory function of the toehold switch. By contrast, the ON/OFF ratios with miR-191 and miR-223 are 1.09 and 1.31, respectively. These ratios are close to 1, meaning the zr146_A toehold switch has high specificity. As a result, zr146_A_ToeholdSwitch-Regulated Invertase has been proven to be useful for miR-146 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-146 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.

Figure. 3. Glucose production under different amounts of miR-146.

Results
As shown above, the glucose concentration rose as the miR-146 triggers increased, representing a positive correlation.

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 40
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 40
    Illegal NheI site found at 1427
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 40
    Illegal BglII site found at 1198
    Illegal BamHI site found at 1328
    Illegal XhoI site found at 1399
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 40
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 40
    Illegal AgeI site found at 999
  • 1000
    COMPATIBLE WITH RFC[1000]