Difference between revisions of "Part:BBa K4207016"

Latest revision as of 12:13, 10 October 2022

BYDV toehold switch B1

1. Usage and Biology

Toehold switches are engineered riboregulators that control the expression of a downstream protein coding sequence. They can be designed to detect virtually any sequence. Toehold switches are designed in silico so that they fold into a pre-determined secondary structure. This structure contains a stable stem-loop that sequesters the ribosome binding site (RBS) and the start codon, thus preventing translation. After a specific trigger RNA binds to the binding site of the toehold, the lower part of the stem-loop unfolds, revealing the start codon. A weak stem remains, but this structure unfolds upon ribosome binding to the RBS, starting translation (Green et al., 2017). This toehold switch was designed to detect conserved sequences in the Barley Yellow Dwarf Virus genome. The structural change of the toehold switch is illustrated in Figure 1.

Figure 1. Toehold switch mechanism. This animation illustrates the operation of the toehold switch. Initially, the structure is in an inactive state and the RBS and the start codon are hidden in the stem-loop. When a specific trigger binds to the binding site, the stem-loop structure opens and the ribosome binding site and start codon are revealed.

To use this toehold switch, it should be assembled in a construct containing a promoter, the toehold switch, a protein-coding sequence, and optionally a terminator if the sensor is not to be used as linear. To prevent frame-shifting, the toehold switch should be combined with the coding sequence using scarless assembly.

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]

2. Design

This toehold switch was designed according to the B-series ideal structure from Pardee et al. (2016). This structure was improved from the original toehold switch structure (Green et al., 2014) to reduce translational leakage. We screened the Barley Yellow Dwarf Virus genome for conserved sequences. Each sequence was divided into 36-nucleotide long subsequences and we designed toehold switches designed to specifically bind to the sequence. This toehold switch was designed using the 21-nucleotide linker (Green et al., 2014). We assigned a score for each toehold switch based on the three-parameter fit from Ma et al. (2018) and selected the best-ranking toehold switches for our library.

3. Characterization

Score predicted by our model: 23,10

Figure 2. Structure of inactive form. The picture was created with the NUPACK web app (Zadeh et al., 2010)
Figure 3. Structure of active form. The picture was created with the NUPACK web app (Zadeh et al., 2011)

The expected structures for inactive and active toehold switches were modeled by using NUPACK’s analysis tool (Zadeh et al., 2011). This toehold switch’s inactive structure does not fully correspond to the ideal structure. Therefore, the ability to exhibit trigger-dependent translation is uncertain. Due to the aforementioned factors, the toehold switch may not be as functional as desired.

4. Conclusion

Based on the data provided by our model as well as the visualization of the structures, conclusions about this toehold switches functionality can not be drawn. This toehold switch could be functional in a sensor construct, but more promising candidates could be designed with more optimized design algorithms. Verifying the functionality is necessary to create a viable toehold sensor construct.

5. References

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

Green, A., Silver, P., Collins, 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

Fornace, M. E., Porubsky, N. J., & Pierce, N. A. (2020). A Unified Dynamic Programming Framework for the Analysis of Interacting Nucleic Acid Strands: Enhanced Models, Scalability, and Speed. ACS Synthetic Biology, 9(10), 2665–2678. https://doi.org/10.1021/acssynbio.9b00523

Ma, D., Shen, L., Wu, K., Diehnelt, C. W., & Green, A. A. (2018). Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. Synthetic Biology, 3(1). https://doi.org/10.1093/synbio/ysy018

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

Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R., Dirks, R. M., & Pierce, N. A. (2010, November 17). NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry, 32(1), 170–173. https://doi.org/10.1002/jcc.21596

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4207016 short</partinfo>
-Toehold switch for the detection of BYDV gRNA
 ===1. Usage and Biology===
+Toehold switches are engineered riboregulators that control the expression of a downstream protein coding sequence. They can be designed to detect virtually any sequence. Toehold switches are designed <i>in silico</i> so that they fold into a pre-determined secondary structure. This structure contains a stable stem-loop that sequesters the ribosome binding site (RBS) and the start codon, thus preventing translation. After a specific trigger RNA binds to the binding site of the toehold, the lower part of the stem-loop unfolds, revealing the start codon. A weak stem remains, but this structure unfolds upon ribosome binding to the RBS, starting translation (Green et al., 2017). This toehold switch was designed to detect conserved sequences in the Barley Yellow Dwarf Virus genome. The structural change of the toehold switch is illustrated in Figure 1.
 <html>
@@ Line 14: / Line 15: @@
 ]]></style>
 <g><g><g><g id="eOrKQUqjyvE6_to" transform="translate(230,115)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE7_to" transform="translate(230,125)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE8_to" transform="translate(230,135)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE9_to" transform="translate(230,145)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE10_to" transform="translate(230,155)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE11_to" transform="translate(230,165)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE12_to" transform="translate(230,175)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE13_to" transform="translate(230,185)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g></g><g id="eOrKQUqjyvE14_to" transform="translate(340,195)"><g transform="translate(-340,-195)"><circle r="5" transform="translate(230 195)" fill="#482564"/><circle r="5" transform="translate(240 195)" fill="#482564"/><circle r="5" transform="translate(250 195)" fill="#482564"/><circle r="5" transform="translate(260 195)" fill="#482564"/><circle r="5" transform="translate(270 195)" fill="#482564"/><circle r="5" transform="translate(280 195)" fill="#482564"/><circle r="5" transform="translate(290 195)" fill="#482564"/><circle r="5" transform="translate(300 195)" fill="#482564"/><circle r="5" transform="translate(310 195)" fill="#482564"/><circle r="5" transform="translate(320 195)" fill="#482564"/><circle r="5" transform="translate(330 195)" fill="#482564"/><circle r="5" transform="translate(340 195)" fill="#482564"/><circle r="5" transform="translate(350 195)" fill="#482564"/><circle r="5" transform="translate(360 195)" fill="#482564"/><circle r="5" transform="translate(370 195)" fill="#482564"/><circle r="5" transform="translate(380 195)" fill="#482564"/><circle r="5" transform="translate(390 195)" fill="#482564"/><circle r="5" transform="translate(400 195)" fill="#482564"/><circle r="5" transform="translate(410 195)" fill="#482564"/><circle r="5" transform="translate(420 195)" fill="#482564"/><circle r="5" transform="translate(430 195)" fill="#482564"/><circle r="5" transform="translate(440 195)" fill="#482564"/><circle r="5" transform="translate(450 195)" fill="#482564"/></g></g></g><g><g><g id="eOrKQUqjyvE40_to" transform="translate(205,110)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE41_to" transform="translate(200,100)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g><g id="eOrKQUqjyvE42_to" transform="translate(205,90)"><circle r="5" transform="translate(0,0)" fill="#482564"/></g></g><g id="eOrKQUqjyvE43_to" transform="translate(222.5,45)"><g transform="translate(-222.5,-45)"><g><circle r="5" transform="translate(205 40)" fill="#482564"/><circle r="5" transform="translate(200 30)" fill="#482564"/><circle r="5" transform="translate(201 19)" fill="#482564"/><g><circle r="5" transform="translate(207 10)" fill="#838383"/><circle r="5" transform="translate(217 5)" fill="#838383"/><circle r="5" transform="translate(228 5)" fill="#838383"/><circle r="5" transform="translate(238 10)" fill="#838383"/><circle r="5" transform="translate(244 19)" fill="#838383"/><circle r="5" transform="translate(245 30)" fill="#838383"/><circle r="5" transform="translate(240 40)" fill="#838383"/></g></g><g><circle r="5" transform="translate(215 85)" fill="#482564"/><circle r="5" transform="translate(215 75)" fill="#482564"/><circle r="5" transform="translate(215 65)" fill="#482564"/><circle r="5" transform="translate(215 55)" fill="#482564"/><circle r="5" transform="translate(215 45)" fill="#482564"/><circle r="5" transform="translate(230 55)" fill="#482564"/><circle r="5" transform="translate(230 45)" fill="#482564"/><circle r="5" transform="translate(230 65)" fill="#482564"/><circle r="5" transform="translate(230 75)" fill="#482564"/><circle r="5" transform="translate(230 85)" fill="#482564"/></g></g></g><g><g id="eOrKQUqjyvE68_to" transform="translate(240,90)"><circle r="5" transform="translate(0,0)" fill="#7f6494"/></g><g id="eOrKQUqjyvE69_to" transform="translate(245,100)"><circle r="5" transform="translate(0,0)" fill="#7f6494"/></g><g id="eOrKQUqjyvE70_to" transform="translate(240,110)"><circle r="5" transform="translate(0,0)" fill="#7f6494"/></g></g></g><g><g><circle r="5" transform="translate(15 195)" fill="#7acbbc"/><circle r="5" transform="translate(35 195)" fill="#7acbbc"/><circle r="5" transform="translate(55 195)" fill="#7acbbc"/><circle r="5" transform="translate(75 195)" fill="#7acbbc"/><circle r="5" transform="translate(95 195)" fill="#7acbbc"/><circle r="5" transform="translate(115 195)" fill="#7acbbc"/><circle r="5" transform="translate(135 195)" fill="#7acbbc"/><circle r="5" transform="translate(155 195)" fill="#7acbbc"/><circle r="5" transform="translate(175 195)" fill="#7acbbc"/><circle r="5" transform="translate(195 195)" fill="#7acbbc"/><circle r="5" transform="translate(215 195)" fill="#7acbbc"/><circle r="5" transform="translate(5 195)" fill="#7acbbc"/><circle r="5" transform="translate(25 195)" fill="#7acbbc"/><circle r="5" transform="translate(45 195)" fill="#7acbbc"/><circle r="5" transform="translate(65 195)" fill="#7acbbc"/><circle r="5" transform="translate(85 195)" fill="#7acbbc"/><circle r="5" transform="translate(105 195)" fill="#7acbbc"/><circle r="5" transform="translate(125 195)" fill="#7acbbc"/><circle r="5" transform="translate(145 195)" fill="#7acbbc"/><circle r="5" transform="translate(165 195)" fill="#7acbbc"/><circle r="5" transform="translate(185 195)" fill="#7acbbc"/><circle r="5" transform="translate(205 195)" fill="#7acbbc"/><g><g id="eOrKQUqjyvE96_to" transform="translate(215,175)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g><g id="eOrKQUqjyvE97_to" transform="translate(215,165)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g><g id="eOrKQUqjyvE98_to" transform="translate(215,155)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g><g id="eOrKQUqjyvE99_to" transform="translate(215,145)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g><g id="eOrKQUqjyvE100_to" transform="translate(215,135)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g><g id="eOrKQUqjyvE101_to" transform="translate(215,125)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g><g id="eOrKQUqjyvE102_to" transform="translate(215,115)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g><g id="eOrKQUqjyvE103_to" transform="translate(215,185)"><circle r="5" transform="translate(0,0)" fill="#7acbbc"/></g></g></g></g><g id="eOrKQUqjyvE104_to" transform="translate(150,220)"><g transform="translate(-150,-210)"><circle r="5" transform="translate(15 210)" fill="#1f8875"/><circle r="5" transform="translate(35 210)" fill="#1f8875"/><circle r="5" transform="translate(55 210)" fill="#1f8875"/><circle r="5" transform="translate(75 210)" fill="#1f8875"/><circle r="5" transform="translate(95 210)" fill="#1f8875"/><circle r="5" transform="translate(115 210)" fill="#1f8875"/><circle r="5" transform="translate(135 210)" fill="#1f8875"/><circle r="5" transform="translate(155 210)" fill="#1f8875"/><circle r="5" transform="translate(175 210)" fill="#1f8875"/><circle r="5" transform="translate(195 210)" fill="#1f8875"/><circle r="5" transform="translate(215 210)" fill="#1f8875"/><circle r="5" transform="translate(225 210)" fill="#1f8875"/><circle r="5" transform="translate(235 210)" fill="#1f8875"/><circle r="5" transform="translate(245 210)" fill="#1f8875"/><circle r="5" transform="translate(255 210)" fill="#1f8875"/><circle r="5" transform="translate(265 210)" fill="#1f8875"/><circle r="5" transform="translate(275 210)" fill="#1f8875"/><circle r="5" transform="translate(285 210)" fill="#1f8875"/><circle r="5" transform="translate(295 210)" fill="#1f8875"/><circle r="5" transform="translate(5 210)" fill="#1f8875"/><circle r="5" transform="translate(25 210)" fill="#1f8875"/><circle r="5" transform="translate(45 210)" fill="#1f8875"/><circle r="5" transform="translate(65 210)" fill="#1f8875"/><circle r="5" transform="translate(85 210)" fill="#1f8875"/><circle r="5" transform="translate(105 210)" fill="#1f8875"/><circle r="5" transform="translate(125 210)" fill="#1f8875"/><circle r="5" transform="translate(145 210)" fill="#1f8875"/><circle r="5" transform="translate(165 210)" fill="#1f8875"/><circle r="5" transform="translate(185 210)" fill="#1f8875"/><circle r="5" transform="translate(205 210)" fill="#1f8875"/></g></g></g></svg>
+<b>Figure 1</b>. Toehold switch mechanism. This animation illustrates the operation of the toehold switch. Initially, the structure is in an inactive state and the RBS and the start codon are hidden in the stem-loop. When a specific trigger binds to the binding site, the stem-loop structure opens and the ribosome binding site and start codon are revealed.
 </div>
 </center>
 </html>
+To use this toehold switch, it should be assembled in a construct containing a promoter, the toehold switch, a protein-coding sequence, and optionally a terminator if the sensor is not to be used as linear. To prevent frame-shifting, the toehold switch should be combined with the coding sequence using scarless assembly.
 <!-- -->
@@ Line 24: / Line 29: @@
 ===2. Design===
+This toehold switch was designed according to the B-series ideal structure from Pardee et al. (2016). This structure was improved from the original toehold switch structure (Green et al., 2014) to reduce translational leakage. We screened the Barley Yellow Dwarf Virus genome for conserved sequences. Each sequence was divided into 36-nucleotide long subsequences and we designed toehold switches designed to specifically bind to the sequence. This toehold switch was designed using the 21-nucleotide linker (Green et al., 2014). We assigned a score for each toehold switch based on the three-parameter fit from Ma et al. (2018) and selected the best-ranking toehold switches for our library.
 ===3. Characterization===
-<gallery caption="Expected structures" widths="400px" heights="400px" perrow="6">
+<b>Score predicted by our model: 23,10</b>
-File:BYDV_B1_inactive.png|Structure of inactive form
+<gallery caption="" widths="400px" heights="400px" perrow="6">
-File:BYDV_B1_active.png|Structure of active form
+File:BYDV_B1_inactive.png|<b>Figure 2.</b> Structure of inactive form. The picture was created with the NUPACK web app (Zadeh et al., 2010)
+File:BYDV_B1_active.png|<b>Figure 3.</b> Structure of active form. The picture was created with the NUPACK web app (Zadeh et al., 2011)
 </gallery>
+The expected structures for inactive and active toehold switches were modeled by using NUPACK’s analysis tool (Zadeh et al., 2011). This toehold switch’s inactive structure does not fully correspond to the ideal structure. Therefore, the ability to exhibit trigger-dependent translation is uncertain. Due to the aforementioned factors, the toehold switch may not be as functional as desired.
 ===4. Conclusion===
+Based on the data provided by our model as well as the visualization of the structures, conclusions about this toehold switches functionality can not be drawn. This toehold switch could be functional in a sensor construct, but more promising candidates could be designed with more optimized design algorithms. Verifying the functionality is necessary to create a viable toehold sensor construct.
 ===5. References===
+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
+Green, A., Silver, P., Collins, 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
+Fornace, M. E., Porubsky, N. J., & Pierce, N. A. (2020). A Unified Dynamic Programming Framework for the Analysis of Interacting Nucleic Acid Strands: Enhanced Models, Scalability, and Speed. ACS Synthetic Biology, 9(10), 2665–2678. https://doi.org/10.1021/acssynbio.9b00523
+Ma, D., Shen, L., Wu, K., Diehnelt, C. W., & Green, A. A. (2018). Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. Synthetic Biology, 3(1). https://doi.org/10.1093/synbio/ysy018
+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
+Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R., Dirks, R. M., & Pierce, N. A. (2010, November 17). NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry, 32(1), 170–173. https://doi.org/10.1002/jcc.21596
 <!-- Uncomment this to enable Functional Parameter display