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Part:BBa_K3454036:Design

Designed by: Ruijie Xiang   Group: iGEM20_ShanghaiTech_China   (2020-10-22)
Revision as of 11:02, 26 October 2020 by Richard Xiang (Talk | contribs)


Activator


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]

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We designed two aptamers containing the structure-switching sequence to lock another oligonucleotide called activator DNA. Since it is locked, it cannot activate Cas12a’s cleavage. But once aptamers have recognized antibiotics, the activators will be released and can then then serve as the target DNA to activate Cas12a. Then as we introduced in <a href = "https://2020.igem.org/Team:ShanghaiTech_China/Proof_Of_Concept">Proof of Concept</a>, the Cas12a will cleave reporter ssDNA and let the whole system emit fluorescence.

 

In fact, several considerations are involved in the designing process of aptamers and activator DNA since they are not exactly the same as the original aptamer. After referring to the literature1 and combining the knowledge we have learned, we summarized the following tips. First and foremost, the structure-switching sequences should be included in the aptamers. Secondly, additional sequences should be added at the front and end of the original aptamer to bind the activator DNA more tightly, which can help reduce leakage. Also, based on the knowledge we knew about Cas12a at that time, the activator should be a double-strand DNA with PAM sequence.

 

However, if the activator is double strand structure, it will be impossible for it to pair with aptamers. How to fix this problem? We returned to the article, and found that the author used single-strand DNA as activator! We did not know who was wrong there. But based on our experience of Cas12a, we did not believe that at first. So, we lay that over and turn to other possible solutions. After several weeks, we did not make any progress in this problem. At the time we almost give up this design, we found out that when Cas12a targets ssDNA, it actually does not need a PAM sequence2! This information dispelled our doubts and boosted our confidence in the design of the aptamers and the activator.

 

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      <img class="developer-image" style="margin-left:0%; width:100%;" src="T--ShanghaiTech_China--contribution1.jpg">

Figure 1.The design norm of locked activator and modifying aptamer.

 

So, we redesigned the element like Figure 1. It obeys the two rules we mentioned above. The structure-switching sequences should be included in the aptamers. And additional sequences should be added at the front and end of the original aptamer to bind the activator DNA more tightly, which can help reduce leakage. And we named it as Aptamer Sandwich. 


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Reference

1.Xiong, Y., Zhang, J., Yang, Z., Mou, Q., Ma, Y., Xiong, Y., & Lu, Y. (2020). Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. Journal of the American Chemical Society, 142(1), 207–213.
<a href = “https://doi.org/10.1021/jacs.9b09211”>https://doi.org/10.1021/jacs.9b09211</a> 

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2.Kellner, M. J., Koob, J. G., & Gootenberg, J. S. (2019). SHERLOCK : nucleic acid detection with CRISPR nucleases. Nature Protocols, 14(October).
<a href = “https://doi.org/10.1038/s41596-019-0210-2”>https://doi.org/10.1038/s41596-019-0210-2</a> 

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Design Notes

Source

It does not come from genomic DNA. It is artificially synthesized.

References