Difference between revisions of "Part:BBa K3431016"
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<partinfo>BBa_K3431016 short</partinfo>
 
<partinfo>BBa_K3431016 short</partinfo>
  
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===Description===
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===Introduction===
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Toehold Switch with Invertase as Expression Protein (pp21_B) is an RNA-based device that is to apply as a biosensor for miRNA. This biosensor is designed to detect and reflect the amount of miRNA-21 through the expression of Beta-Fructosidase (Thermotoga Maritima MSB8) (BBa_K3431000), which can convert sucrose into glucose in a time-saving process and its result can be easily represented in the readout of glucose meter. The mechanism for detection relied on the following part - Toehold Switch for miRNA-21 (pp21_B) (BBa_K3431001) - whose restriction on the expression of invertase can be liberated upon binding with miRNA-21. Furthermore, we include T7 promoter  (BBa_I719005) and terminator (BBa_K731721) to insure that our device can transcribe and translate in the environment of PURExpress in vitro protein synthesis kit.
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pp21_B_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-21 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).
  
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===Experiment result===
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===Components===
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pp21_B_ToeholdSwitch-Regulated Invertase consists of 4 basic parts: T7 promoter (BBa_I719005), pp21_B toehold switch (BBa_K3431001), invertase (BBa_K3431000), and T7 terminator (BBa_K731721).
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The mechanism of the detection is mainly based on the regulatory part, pp21_B Toehold Switch for miR-21 Detection (BBa_K3431001). Upon binding with miR-21, 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).
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As for the T7 promoter (BBa_I719005) and T7 terminator (BBa_K731721), they are the essential genetic elements for the PURExpress protein synthesis kit.
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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.
 
<html>
 
<html>
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<div style="width=100%; display:flex; align-items: center; justify-content: center">
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<img src="https://static.igem.org/mediawiki/parts/3/37/T--CSMU_Taiwan--Fig._6_%28In_fusion_cloning%29.png" style="width:50%">
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</div>
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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.
 
<br>
 
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</html>
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===Response in different miRNA===
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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”.
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<html>
 
<div style="width=100%; display:flex; align-items: center; justify-content: center">
 
<div style="width=100%; display:flex; align-items: center; justify-content: center">
 
<img src="https://static.igem.org/mediawiki/parts/e/e5/T--CSMU_Taiwan--pp21_B_%28BBa_K3431001%29.png" style="width:50%">
 
<img src="https://static.igem.org/mediawiki/parts/e/e5/T--CSMU_Taiwan--pp21_B_%28BBa_K3431001%29.png" style="width:50%">
 
</div>
 
</div>
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Figure. 2. The glucose productions of the pp21_B_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-21 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).
 
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</html>
 
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<br>
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<b>Results</b><br>
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The ON/OFF ratio with miR-21 is 0.96, which suggested the leakage problem. The regulatory function of the pp21_B toehold switch was not good enough. Thus, pp21_B toehold switch-regulated invertase cannot be well controlled by the miR-21.
  
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===References===
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===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.
 
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.
 
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.
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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
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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.
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3431016 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3431021 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3431016 parameters</partinfo>
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<partinfo>BBa_K3431021 parameters</partinfo>
 
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Revision as of 17:15, 25 October 2020


pp21_B_ToeholdSwitch-Regulated Invertase

Introduction

pp21_B_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-21 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

pp21_B_ToeholdSwitch-Regulated Invertase consists of 4 basic parts: T7 promoter (BBa_I719005), pp21_B toehold switch (BBa_K3431001), invertase (BBa_K3431000), and T7 terminator (BBa_K731721). The mechanism of the detection is mainly based on the regulatory part, pp21_B Toehold Switch for miR-21 Detection (BBa_K3431001). Upon binding with miR-21, 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 2020 iGEM CSMU-Taiwan used in-fusion cloning to construct the composite part. The process is shown below.

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.

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 pp21_B_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-21 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 ON/OFF ratio with miR-21 is 0.96, which suggested the leakage problem. The regulatory function of the pp21_B toehold switch was not good enough. Thus, pp21_B toehold switch-regulated invertase cannot be well controlled by the miR-21.

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
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1425
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1196
    Illegal BamHI site found at 1326
    Illegal XhoI site found at 1397
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 997
  • 1000
    COMPATIBLE WITH RFC[1000]