Difference between revisions of "Part:BBa K3431001"

Latest revision as of 11:56, 9 October 2021

pp21_B Toehold Switch for miR-21 Detection

Introduction

pp21_B toehold switch is a regulatory part for the downstream reporter gene. With this part, the protein expression can be controlled by the miR-21. The sequence of the toehold switch can be separated into the following 5 regions from its 5' end: TBS (trigger binding site), stem region, loop region with RBS (ribosome binding site), complimentary stem region with a start codon, and linker. 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 of the downstream reporter.

Design

The design of the toehold switch was mainly based on the previous research^{[1][2][3][4][5][6]}. For the pp21_B toehold switch, we adopted the loop and the linker structure from Wang et al., 2019^[7]. Using NUPACK analysis and Vienna binding models, we designed the sequence of the toehold switch. (See our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model )

Characterization using invertase

The 2020 iGEM CSMU-Taiwan characterized the toehold switch with invertase (BBa_K3431000) reporter protein. 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. 3. The glucose productions of the pp21_B 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-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.

References

1. 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. https://doi.org/10.1016/j.cell.2014.10.002

2. Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. Nature, 548(7665), 117–121. https://doi.org/10.1038/nature23271

3. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O'Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. https://doi.org/10.1016/j.cell.2016.04.059

4. Chappell, J., Westbrook, A., Verosloff, M., & Lucks, J. B. (2017). Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nature communications, 8(1), 1051. https://doi.org/10.1038/s41467-017-01082-6

5. Sadat Mousavi, P., Smith, S. J., Chen, J. B., Karlikow, M., Tinafar, A., Robinson, C., Liu, W., Ma, D., Green, A. A., Kelley, S. O., & Pardee, K. (2020). A multiplexed, electrochemical interface for gene-circuit-based sensors. Nature chemistry, 12(1), 48–55. https://doi.org/10.1038/s41557-019-0366-y

6. Hong, F., Ma, D., Wu, K., Mina, L. A., Luiten, R. C., Liu, Y., Yan, H., & Green, A. A. (2020). Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators. Cell, 180(5), 1018–1032.e16. https://doi.org/10.1016/j.cell.2020.02.011

7. Shue Wang, Nicholas J Emery, Allen P Liu. A Novel Synthetic Toehold Switch for microRNA Detection in Mammalian Cells. ACS Synthetic Biology 2019; 8 (5): 1079-1088.

Information contributed by City of London UK (2021)

This toehold switch was characterized in silico using the ToeholdTools project that our team developed. See https://github.com/lkn849/thtools for more information.

Metadata:

Group: City of London UK 2021
Author: Lucas Ng
Summary: Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.

Raw data:

This contribution was autogenerated by the script contrib.py, available at https://github.com/lkn849/thtools/tree/master/registry.

This switch was designed to detect the miRNA hsa-miR-21-5p at a temperature of 37°C. We tested it against every mature Homo sapiens miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-7156-5p.

With hsa-miR-7156-5p at 37°C, the switch has a specificity of 14 ± Infinity % and an activation of 0.2 ± 0.9 %. These values represent 95% confidence limits (z=1.96).

The temperature–activation–specificity relationship is shown here. CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:

Error bars represent the standard deviation. The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.

Caveats:

As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.
The miRNA most targeted by this switch heavily fluctuates based on temperature.Therefore, we cannot confirm the reliability of this switch.

We do not recommend this part for future usage. 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]

@@ Line 45: / Line 45: @@
 <b>Results</b><br>
 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.
+===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. https://doi.org/10.1016/j.cell.2014.10.002
+. Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. Nature, 548(7665), 117–121. https://doi.org/10.1038/nature23271
+. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O'Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. https://doi.org/10.1016/j.cell.2016.04.059
+. Chappell, J., Westbrook, A., Verosloff, M., & Lucks, J. B. (2017). Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nature communications, 8(1), 1051. https://doi.org/10.1038/s41467-017-01082-6
+. Sadat Mousavi, P., Smith, S. J., Chen, J. B., Karlikow, M., Tinafar, A., Robinson, C., Liu, W., Ma, D., Green, A. A., Kelley, S. O., & Pardee, K. (2020). A multiplexed, electrochemical interface for gene-circuit-based sensors. Nature chemistry, 12(1), 48–55. https://doi.org/10.1038/s41557-019-0366-y
+. Hong, F., Ma, D., Wu, K., Mina, L. A., Luiten, R. C., Liu, Y., Yan, H., & Green, A. A. (2020). Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators. Cell, 180(5), 1018–1032.e16. https://doi.org/10.1016/j.cell.2020.02.011
+. Shue Wang, Nicholas J Emery, Allen P Liu. A Novel Synthetic Toehold Switch for microRNA Detection in Mammalian Cells. ACS Synthetic Biology 2019; 8 (5): 1079-1088.
 ===Information contributed by City of London UK (2021)===
@@ Line 61: / Line 75: @@
 *[[Media:BBa_K3431001_crt.txt]]
-This contribution was autogenerated by the script https://github.com/lkn849/thtools/registry/contrib.py
+This contribution was autogenerated by the script '''contrib.py''', available at https://github.com/lkn849/thtools/tree/master/registry.
 ----
-This switch was designed to detect the miRNA hsa-miR-21-5p at a temperature of 37.
+This switch was designed to detect the miRNA hsa-miR-21-5p at a temperature of 37°C.
-We tested it against every <i>Homo sapiens</i> RNA in miRBase and our analysis shows that it is best used to detect hsa-miR-4729.
+We tested it against every mature <i>Homo sapiens</i> miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-7156-5p.
-With hsa-miR-4729 at 37°C, the switch has a specificity of 11 ± Infinity % and an activation of 0.2 ± 0.5 %.
+With hsa-miR-7156-5p at 37°C, the switch has a specificity of 14 ± Infinity % and an activation of 0.2 ± 0.9 %.
 These values represent 95% confidence limits (z=1.96).
-The temperature&ndash;activation&ndash;specificity relationship is shown here:
+The temperature&ndash;activation&ndash;specificity relationship is shown here.
+CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:
-[[File:BBa_K3431001_crt.png|500px|center]]
+[[File:BBa_K3431001_graph.png|500px|center]]
-Error bars represent the standard error (SE).
+Error bars represent the standard deviation.
-The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's SE.
+The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.
 '''Caveats:'''
-*As per the above, we cannot confirm that this switch detects the desired RNA sequence.
+*As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.
+*The miRNA most targeted by this switch heavily fluctuates based on temperature.Therefore, we cannot confirm the reliability of this switch.
 We do not recommend this part for future usage.
-===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. https://doi.org/10.1016/j.cell.2014.10.002
-. Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. Nature, 548(7665), 117–121. https://doi.org/10.1038/nature23271
-. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O'Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. https://doi.org/10.1016/j.cell.2016.04.059
-. Chappell, J., Westbrook, A., Verosloff, M., & Lucks, J. B. (2017). Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nature communications, 8(1), 1051. https://doi.org/10.1038/s41467-017-01082-6
-. Sadat Mousavi, P., Smith, S. J., Chen, J. B., Karlikow, M., Tinafar, A., Robinson, C., Liu, W., Ma, D., Green, A. A., Kelley, S. O., & Pardee, K. (2020). A multiplexed, electrochemical interface for gene-circuit-based sensors. Nature chemistry, 12(1), 48–55. https://doi.org/10.1038/s41557-019-0366-y
-. Hong, F., Ma, D., Wu, K., Mina, L. A., Luiten, R. C., Liu, Y., Yan, H., & Green, A. A. (2020). Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators. Cell, 180(5), 1018–1032.e16. https://doi.org/10.1016/j.cell.2020.02.011
-. Shue Wang, Nicholas J Emery, Allen P Liu. A Novel Synthetic Toehold Switch for microRNA Detection in Mammalian Cells. ACS Synthetic Biology 2019; 8 (5): 1079-1088.
 <!-- Add more about the biology of this part here
 ===Usage and Biology===