Difference between revisions of "Part:BBa K4245160:Design"
 
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===Design Notes===
 
===Design Notes===
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RCT yields long strands of repeating RNA sequences; to prevent the misfolding of the transcribed aptamers, it was necessary to include an upstream spacer sequence, a method inspired by the inclusion of a spacer sequence between two DNA aptamers for RCA done by researchers at the University of Arkansas (Al-Ogaili et al., 2020). The padlock spacer the team selected was derived from the Green Linker (Green, Silver, Collins, & Yin, 2014). We retrieved the Green Linker from the literature and manually modified it in NUPACK until there was no folding at our desired temperature of 25°C (Takahashi et. al, 2018) (see Fig. 2). We then added this sequence upstream of the Broccoli complement sequence in our padlock probe.  
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RCT yields long strands of repeating RNA sequences; to prevent the misfolding of the transcribed aptamers, it was necessary to include an upstream spacer sequence, a method inspired by the inclusion of a spacer sequence between two DNA aptamers for RCA done by researchers at the University of Arkansas (Al-Ogaili et al., 2020). The padlock spacer the team selected was derived from the Green Linker (Green, Silver, Collins, & Yin, 2014). We retrieved the Green Linker from the literature and manually modified it in NUPACK until there was no folding at our desired temperature of 25°C (Takahashi et. al, 2018) (see Fig. 1). We then added this sequence upstream of the Broccoli complement sequence in our padlock probe.  
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[[File:BBa_K4245160_Spacer_Predicted_Folding_Snapgene.jpeg|thumb|center|500px|<i>Figure 2.  The folding of the padlock spacer sequence, BBa_K4245160, in Snapgene.</i>]]
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[[File:BBa_K4245160_Spacer_Predicted_Folding_Snapgene.jpeg|thumb|center|500px|<i>Figure 1.  The folding of the padlock spacer sequence, BBa_K4245160, in Snapgene.</i>]]
 
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Takahashi, M. K., Tan, X., Dy, A. J., Braff, D., Akana, R. T., Furuta, Y., Donghia, N., Ananthakrishnan, A., & Collins, J. J. (2018). A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-05864-4
 
Takahashi, M. K., Tan, X., Dy, A. J., Braff, D., Akana, R. T., Furuta, Y., Donghia, N., Ananthakrishnan, A., & Collins, J. J. (2018). A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-05864-4
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Latest revision as of 12:58, 10 October 2022


Padlock spacer sequence


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]


Design Notes

RCT yields long strands of repeating RNA sequences; to prevent the misfolding of the transcribed aptamers, it was necessary to include an upstream spacer sequence, a method inspired by the inclusion of a spacer sequence between two DNA aptamers for RCA done by researchers at the University of Arkansas (Al-Ogaili et al., 2020). The padlock spacer the team selected was derived from the Green Linker (Green, Silver, Collins, & Yin, 2014). We retrieved the Green Linker from the literature and manually modified it in NUPACK until there was no folding at our desired temperature of 25°C (Takahashi et. al, 2018) (see Fig. 1). We then added this sequence upstream of the Broccoli complement sequence in our padlock probe.

Figure 1. The folding of the padlock spacer sequence, BBa_K4245160, in Snapgene.


Takahashi, M. K., Tan, X., Dy, A. J., Braff, D., Akana, R. T., Furuta, Y., Donghia, N., Ananthakrishnan, A., & Collins, J. J. (2018). A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-05864-4


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