Difference between revisions of "Part:BBa K3714010"
 
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This is an improved version of <html><a href="https://parts.igem.org/Part:BBa_K3714002">BBa_K3714002</a></html>, which is an aptazyme designed for detecting gardiquimod by lateral flow assay (LFA). We aimed to control the cleavage of aptazyme so we can transcribe them in advance to shorten overall time consumption.  
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This is an improved version of <html><a href="https://parts.igem.org/Part:BBa_K3714002">BBa_K3714002</a></html>, which is an aptazyme designed for detecting gardiquimod by lateral flow assay (LFA). We aimed to control the cleavage of aptazyme so we can transcribe them in advance to shorten overall time consumption. <br/>
 
To test our optimized design with TMSD(toehold mediated strand displacement) reaction, a toehold site between biotin probe binding site and aptazyme domain is necessary (Figure.1). However, designing the toehold and probe manually and testing with available RNA structure prediction model is a huge work with consuming much labor. <br/>
 
To test our optimized design with TMSD(toehold mediated strand displacement) reaction, a toehold site between biotin probe binding site and aptazyme domain is necessary (Figure.1). However, designing the toehold and probe manually and testing with available RNA structure prediction model is a huge work with consuming much labor. <br/>
 
<center>[[File:K3714010-0.png]]</center>
 
<center>[[File:K3714010-0.png]]</center>

Latest revision as of 03:46, 22 October 2021


Improved gard-337 for lateral flow assay
This is an improved version of BBa_K3714002, which is an aptazyme designed for detecting gardiquimod by lateral flow assay (LFA). We aimed to control the cleavage of aptazyme so we can transcribe them in advance to shorten overall time consumption.
To test our optimized design with TMSD(toehold mediated strand displacement) reaction, a toehold site between biotin probe binding site and aptazyme domain is necessary (Figure.1). However, designing the toehold and probe manually and testing with available RNA structure prediction model is a huge work with consuming much labor.

K3714010-0.png
Figure.1 | Design of temporarily inhibition and disinhibition by oligonucleotides.

The toehold site in part was designed by our probe design model and verified by the prediction model of team: SJTU-Software. And the changed sequences shows no influence on the function of aptazyme(Figure.2, also see the unchanged BBa_K3714002), the toehold worked as our expect as well (Figure.2).
The stop oligo significantly increased the percentage of the uncleaved band, resembling the effect of gardiquimod (the inhibitor of this aptamer). Thus our engineering is successful.

K3714016.png
Figure.2 | Verification of temporarily inhibition and disinhibition by oligonucleotides. The aptazyme was produced by a T7 in vitro transcription system. The product was run on a denatured PAGE. Gardiquimod is an inhibitor of the cleavage activity and "-gardiquimod" is a control group. "stop" and "start" are two oligonucleotides designed to control the activity of the aptazyme.

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]