Difference between revisions of "Part:BBa K4207044"

 
 
(2 intermediate revisions by the same user not shown)
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
<partinfo>BBa_K4207043 short</partinfo>
+
<partinfo>BBa_K4207044 short</partinfo>
  
Toehold switch for the detection of PMMV gRNA
+
===1. Usage and Biology===
  
<!-- Add more about the biology of this part here
+
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 Pepper mild mottle virus genome. The structural change of the toehold switch is illustrated in Figure 1.
===Usage and Biology===
+
 
 +
<html>
 +
<center>
 +
<div style="width: 60%;">
 +
<svg id="eOrKQUqjyvE1" viewBox="0 0 455 215" shape-rendering="geometricPrecision" text-rendering="geometricPrecision">
 +
<style><![CDATA[
 +
#eOrKQUqjyvE6_to {animation: eOrKQUqjyvE6_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE6_to__to { 0% {transform: translate(230px,115px)} 11.222222% {transform: translate(230px,115px)} 55.666667% {transform: translate(317.272727px,195px)} 72.333333% {transform: translate(390px,195px)} 100% {transform: translate(390px,195px)}} #eOrKQUqjyvE7_to {animation: eOrKQUqjyvE7_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE7_to__to { 0% {transform: translate(230px,125px)} 11.222222% {transform: translate(230px,125px)} 50.111111% {transform: translate(306.363636px,195px)} 72.333333% {transform: translate(400px,195px)} 100% {transform: translate(400px,195px)}} #eOrKQUqjyvE8_to {animation: eOrKQUqjyvE8_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE8_to__to { 0% {transform: translate(230px,135px)} 11.222222% {transform: translate(230px,135px)} 44.444444% {transform: translate(295.454545px,195px)} 72.222222% {transform: translate(410px,195px)} 100% {transform: translate(410px,195px)}} #eOrKQUqjyvE9_to {animation: eOrKQUqjyvE9_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE9_to__to { 0% {transform: translate(230px,145px)} 11.222222% {transform: translate(230px,145px)} 39% {transform: translate(284.545454px,195px)} 72.333333% {transform: translate(420px,195px)} 100% {transform: translate(420px,195px)}} #eOrKQUqjyvE10_to {animation: eOrKQUqjyvE10_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE10_to__to { 0% {transform: translate(230px,155px)} 11.222222% {transform: translate(230px,155px)} 33.444444% {transform: translate(273.636364px,195px)} 72.333333% {transform: translate(430px,195px)} 100% {transform: translate(430px,195px)}} #eOrKQUqjyvE11_to {animation: eOrKQUqjyvE11_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE11_to__to { 0% {transform: translate(230px,165px)} 11.222222% {transform: translate(230px,165px)} 27.888889% {transform: translate(262.727273px,195px)} 72.333333% {transform: translate(440px,195px)} 100% {transform: translate(440px,195px)}} #eOrKQUqjyvE12_to {animation: eOrKQUqjyvE12_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE12_to__to { 0% {transform: translate(230px,175px)} 11.222222% {transform: translate(230px,175px)} 22.333333% {transform: translate(251.818182px,195px)} 72.333333% {transform: translate(450px,195px)} 100% {transform: translate(450px,195px)}} #eOrKQUqjyvE13_to {animation: eOrKQUqjyvE13_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE13_to__to { 0% {transform: translate(230px,185px)} 11.222222% {transform: translate(230px,185px)} 16.777778% {transform: translate(240.909091px,195px)} 72.333333% {transform: translate(460.431893px,195px)} 100% {transform: translate(460.431893px,195px)}} #eOrKQUqjyvE14_to {animation: eOrKQUqjyvE14_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE14_to__to { 0% {transform: translate(340px,195px)} 11.222222% {transform: translate(340px,195px)} 72.333333% {transform: translate(580.863784px,195px)} 100% {transform: translate(580.863784px,195px)}} #eOrKQUqjyvE40_to {animation: eOrKQUqjyvE40_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE40_to__to { 0% {transform: translate(205px,110px)} 11.222222% {transform: translate(205px,110px)} 59% {transform: translate(300px,195px)} 61.222222% {transform: translate(305px,195px)} 100% {transform: translate(305px,195px)}} #eOrKQUqjyvE41_to {animation: eOrKQUqjyvE41_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE41_to__to { 0% {transform: translate(200px,100px)} 11.222222% {transform: translate(200px,100px)} 64.555556% {transform: translate(305.727273px,195px)} 66.777778% {transform: translate(315px,195px)} 100% {transform: translate(315px,195px)}} #eOrKQUqjyvE42_to {animation: eOrKQUqjyvE42_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE42_to__to { 0% {transform: translate(205px,90px)} 11.222222% {transform: translate(205px,90px)} 70.111111% {transform: translate(320.636364px,194.333333px)} 72.333333% {transform: translate(325px,195px)} 100% {transform: translate(325px,195px)}} #eOrKQUqjyvE43_to {animation: eOrKQUqjyvE43_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE43_to__to { 0% {transform: translate(222.5px,45px)} 11.222222% {transform: translate(222.5px,45px)} 72.333333% {transform: translate(342.5px,155px)} 100% {transform: translate(342.5px,155px)}} #eOrKQUqjyvE68_to {animation: eOrKQUqjyvE68_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE68_to__to { 0% {transform: translate(240px,90px)} 11.222222% {transform: translate(240px,90px)} 69% {transform: translate(353.454545px,194px)} 72.333333% {transform: translate(360px,195px)} 100% {transform: translate(360px,195px)}} #eOrKQUqjyvE69_to {animation: eOrKQUqjyvE69_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE69_to__to { 0% {transform: translate(245px,100px)} 11.222222% {transform: translate(245px,100px)} 63.444444% {transform: translate(347.545454px,194px)} 72.333333% {transform: translate(370px,195px)} 100% {transform: translate(370px,195px)}} #eOrKQUqjyvE70_to {animation: eOrKQUqjyvE70_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE70_to__to { 0% {transform: translate(240px,110px)} 11.222222% {transform: translate(240px,110px)} 59% {transform: translate(333.818181px,195px)} 72.333333% {transform: translate(380px,195px)} 100% {transform: translate(380px,195px)}} #eOrKQUqjyvE96_to {animation: eOrKQUqjyvE96_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE96_to__to { 0% {transform: translate(215px,175px)} 11.222222% {transform: translate(215px,175px)} 21.111111% {transform: translate(234.208633px,192.8px)} 22.333333% {transform: translate(235px,195px)} 100% {transform: translate(235px,195px)}} #eOrKQUqjyvE97_to {animation: eOrKQUqjyvE97_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE97_to__to { 0% {transform: translate(215px,165px)} 11.222222% {transform: translate(215px,165px)} 26.666667% {transform: translate(245px,192.8px)} 27.888889% {transform: translate(245px,195px)} 100% {transform: translate(245px,195px)}} #eOrKQUqjyvE98_to {animation: eOrKQUqjyvE98_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE98_to__to { 0% {transform: translate(215px,155px)} 11.222222% {transform: translate(215px,155px)} 31.111111% {transform: translate(254.054545px,190.8px)} 33.444444% {transform: translate(255px,195px)} 100% {transform: translate(255px,195px)}} #eOrKQUqjyvE99_to {animation: eOrKQUqjyvE99_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE99_to__to { 0% {transform: translate(215px,145px)} 11.222222% {transform: translate(215px,145px)} 36.666667% {transform: translate(264.963636px,190.8px)} 39% {transform: translate(265px,195px)} 100% {transform: translate(265px,195px)}} #eOrKQUqjyvE100_to {animation: eOrKQUqjyvE100_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE100_to__to { 0% {transform: translate(215px,135px)} 11.222222% {transform: translate(215px,135px)} 41.111111% {transform: translate(273.690909px,188.8px)} 44.555556% {transform: translate(275px,195px)} 100% {transform: translate(275px,195px)}} #eOrKQUqjyvE101_to {animation: eOrKQUqjyvE101_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE101_to__to { 0% {transform: translate(215px,125px)} 11.222222% {transform: translate(215px,125px)} 46.666667% {transform: translate(284.6px,188.8px)} 50.111111% {transform: translate(285px,195px)} 100% {transform: translate(285px,195px)}} #eOrKQUqjyvE102_to {animation: eOrKQUqjyvE102_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE102_to__to { 0% {transform: translate(215px,115px)} 11.222222% {transform: translate(215px,115px)} 50% {transform: translate(291.145455px,184.8px)} 55.666667% {transform: translate(295px,195px)} 100% {transform: translate(295px,195px)}} #eOrKQUqjyvE103_to {animation: eOrKQUqjyvE103_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE103_to__to { 0% {transform: translate(215px,185px)} 11.222222% {transform: translate(215px,185px)} 16.111111% {transform: translate(224.496403px,193.8px)} 16.777778% {transform: translate(225px,195px)} 100% {transform: translate(225px,195px)}} #eOrKQUqjyvE104_to {animation: eOrKQUqjyvE104_to__to 9000ms linear infinite normal forwards}@keyframes eOrKQUqjyvE104_to__to { 0% {transform: translate(150px,220px);animation-timing-function: cubic-bezier(0,0,0.58,1)} 11.111111% {transform: translate(150px,207px)} 100% {transform: translate(150px,207px)}}
 +
]]></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.
  
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K4207043 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4207044 SequenceAndFeatures</partinfo>
 +
 
 +
===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 Pepper mild mottle 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===
 +
 
 +
<b>Score predicted by our model: 26,59</b>
 +
<gallery caption="" widths="400px" heights="400px" perrow="6">
 +
File:PMMV_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:PMMV_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 switches structure closely resembles the ideal inactive and active structures.
 +
 
 +
===4. Conclusion===
 +
 
 +
Based on the data provided by our model as well as the visualization of the structures, this toehold switch is expected to exhibit trigger-dependent translation. This toehold is likely to be effective at sensing a conserved sequence in Pepper mild mottle virus genome. However, 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  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
<partinfo>BBa_K4207043 parameters</partinfo>
+
<partinfo>BBa_K4207044 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 09:12, 10 October 2022


PMMV 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 Pepper mild mottle 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 Pepper mild mottle 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: 26,59

The expected structures for inactive and active toehold switches were modeled by using NUPACK’s analysis tool (Zadeh et al., 2011). This toehold switches structure closely resembles the ideal inactive and active structures.

4. Conclusion

Based on the data provided by our model as well as the visualization of the structures, this toehold switch is expected to exhibit trigger-dependent translation. This toehold is likely to be effective at sensing a conserved sequence in Pepper mild mottle virus genome. However, 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