Difference between revisions of "Part:BBa K3431005"
(→‎Description)
Line 3: Line 3:
 
<partinfo>BBa_K3431005 short</partinfo>
 
<partinfo>BBa_K3431005 short</partinfo>
  
−
===Description===
+
===Introduction===
−
This toehold switch has been designed to open up its hairpin loop structure upon binding with miRNA-21, resulting in the translation of downstream reporter protein. The design of toehold switch can be separated into the following 5 regions from its 5' end: trigger binding sites, stem region, loop region with RBS, complimentary stem region with start codon, and linker amino acids. In our constructions of toehold switches for miRNA-21, we optimised the loop region with RBS and linker amino acids based on three articles: the original work on toehold switch (Green, A.A. et al., 2014), the adaptation of toehold switch to detect zika virus (Pardee, K. et al., 2016), and novel toehold switch design for detection of miRNA in mammalian cells (Wang, S. et al., 2019) . The loop structure from the article of Pardee, K. et al. has been truncated to 12 base pairs compared to the work from Green, A.A. et al. in order to reduce the leakage of output expression. We chose to test the 3 different loop structures and 2 different linker structures (Pardee, K. et al. and Wang, S. et al.) from the above-mentioned studies.
+
op21_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-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).
  
−
For this particular toehold switch (op21_A), we incorporate the loop structure from Green, A.A. et al. and the linker structure from Wang, S. et al..
+
===Components===
 +
op21_A_ToeholdSwitch-Regulated Invertase consists of 4 basic parts: T7 promoter (BBa_I719005), op21_A toehold switch (BBa_K3431005), invertase(BBa_K3431000), and T7 terminator (BBa_K731721).The mechanism of the detection is mainly based on the regulatory part, op21_A Toehold Switch for miR-21 Detection (BBa_K3431005). 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.
 +
<html>
 +
<br>
 +
<div style="width=100%; display:flex; align-items: center; justify-content: center">
 +
<img src="https://static.igem.org/mediawiki/parts/3/37/T--CSMU_Taiwan--Fig._6_%28In_fusion_cloning%29.png" style="width:50%">
 +
</div>
 +
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>
 +
</html>
  
 
===Model===
 
===Model===

Revision as of 11:49, 25 October 2020


op21_A Toehold Switch for miR-21 Detection

Introduction

op21_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-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

op21_A_ToeholdSwitch-Regulated Invertase consists of 4 basic parts: T7 promoter (BBa_I719005), op21_A toehold switch (BBa_K3431005), invertase(BBa_K3431000), and T7 terminator (BBa_K731721).The mechanism of the detection is mainly based on the regulatory part, op21_A Toehold Switch for miR-21 Detection (BBa_K3431005). 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.

Model


NUPACK ANALYSIS


VIENNA RNA PACKAGE

Link to our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model

Experiment result



References

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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]