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</tr> | </tr> | ||
<tr><td colspan=6 align=center valign=center> | <tr><td colspan=6 align=center valign=center> | ||
− | <h3>Protein | + | <h3>Protein Cloning, Expression and Purification</h3> |
<p> | <p> | ||
We decided to compare three versions of Cas13a that were previously characterized in the literature<sup><a class="myLink" href="#ref_1">1</a></sup>: Lbu, Lsh, and Lwa. We ordered the Lbu and Lsh plasmids from Addgene, and we cloned Lwa using Golden Gate assembly (sequence was taken from Gootenberg et al., 2017). Lbu and Lsh were expressed in <i>E.coli</i> Rosetta2, as the sequences were not codon-optimized, and Lwa was expressed in <i>E.coli</i> BL21 (DE3) star. We created three BioBricks from the Lwa sequence: <a class="myLink" href="http://parts.igem.org/Part:BBa_K2323000">BBa_K2323000</a> (containing the Lwa coding sequence and a Tphi terminator), <a class="myLink" href="http://parts.igem.org/Part:BBa_K2323001">BBa_K2323001</a> (where a 6xHis/Twin strep tag and a SUMO tag are added to the N-terminal end of BBa_K2323000), and <a href="http://parts.igem.org/Part:BBa_K2323004">BBa_K2323004</a> (where BBa_K2323001 is preceded by the T7 promoter and the Elowitz RBS). We improved the TEV-protease <a class="myLink" href="http://parts.igem.org/Part:BBa_K1319008">BBa_K1319008</a> by tagging it with a 6xHis tag, purified it and successfully used it for the TEV cleavage of our Cas13a proteins. </p> | We decided to compare three versions of Cas13a that were previously characterized in the literature<sup><a class="myLink" href="#ref_1">1</a></sup>: Lbu, Lsh, and Lwa. We ordered the Lbu and Lsh plasmids from Addgene, and we cloned Lwa using Golden Gate assembly (sequence was taken from Gootenberg et al., 2017). Lbu and Lsh were expressed in <i>E.coli</i> Rosetta2, as the sequences were not codon-optimized, and Lwa was expressed in <i>E.coli</i> BL21 (DE3) star. We created three BioBricks from the Lwa sequence: <a class="myLink" href="http://parts.igem.org/Part:BBa_K2323000">BBa_K2323000</a> (containing the Lwa coding sequence and a Tphi terminator), <a class="myLink" href="http://parts.igem.org/Part:BBa_K2323001">BBa_K2323001</a> (where a 6xHis/Twin strep tag and a SUMO tag are added to the N-terminal end of BBa_K2323000), and <a href="http://parts.igem.org/Part:BBa_K2323004">BBa_K2323004</a> (where BBa_K2323001 is preceded by the T7 promoter and the Elowitz RBS). We improved the TEV-protease <a class="myLink" href="http://parts.igem.org/Part:BBa_K1319008">BBa_K1319008</a> by tagging it with a 6xHis tag, purified it and successfully used it for the TEV cleavage of our Cas13a proteins. </p> | ||
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<tr><td class="verticalColumn" colspan=3 align=center valign=center> | <tr><td class="verticalColumn" colspan=3 align=center valign=center> | ||
− | <h3 id="Figure_1">Proof-of- | + | <h3 id="Figure_1">Proof-of-Concept</h3> |
<p> | <p> | ||
To prove the functionality of Cas13a, we used the 16S rRNA sequence from <i>E.coli</i> as a target sequence, given that is highly conserved in all bacterial species and can be easily extracted from bacterial cultures in large concentrations. For our first experiments, we used only 130 nucleotides of the 16s rRNA sequence and transcribed <i>in vitro</i> from a DNA template (since the whole 16s rRNA is 1500 nucleotides, therefore too large to be transcribed). Our crRNA DNA template was designed so that the target-binding region could easily be changed to detect new targets[scheme or link]. We found that both Lbu and Lwa were functional and degraded the read-out RNase Alert in presence of both the target and the crRNA. An example time plot is shown in <a class="myLink" href="#Figure_1">Figure 1</a>, where the specific activity of Lbu was controlled by taking out the crRNA and Lbu, alternatively. | To prove the functionality of Cas13a, we used the 16S rRNA sequence from <i>E.coli</i> as a target sequence, given that is highly conserved in all bacterial species and can be easily extracted from bacterial cultures in large concentrations. For our first experiments, we used only 130 nucleotides of the 16s rRNA sequence and transcribed <i>in vitro</i> from a DNA template (since the whole 16s rRNA is 1500 nucleotides, therefore too large to be transcribed). Our crRNA DNA template was designed so that the target-binding region could easily be changed to detect new targets[scheme or link]. We found that both Lbu and Lwa were functional and degraded the read-out RNase Alert in presence of both the target and the crRNA. An example time plot is shown in <a class="myLink" href="#Figure_1">Figure 1</a>, where the specific activity of Lbu was controlled by taking out the crRNA and Lbu, alternatively. | ||
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<tr><td colspan=6 align=center valign=center> | <tr><td colspan=6 align=center valign=center> | ||
− | <h3>Detection of | + | <h3>Detection of Pathogenic RNA from <i>in Vivo</i> Source</h3> |
<p> | <p> | ||
We then set out to detect RNA from <i>in vivo</i> samples rather than from <i>in vitro</i> transcribed RNA. As we had chosen the 16S rRNA sequence of <i>E. coli</i> as a target, we used <i>E. coli</i> DH5α cultures as <i>in vivo</i> samples. We performed two kinds of treatment on the cells (from an overnight culture): | We then set out to detect RNA from <i>in vivo</i> samples rather than from <i>in vitro</i> transcribed RNA. As we had chosen the 16S rRNA sequence of <i>E. coli</i> as a target, we used <i>E. coli</i> DH5α cultures as <i>in vivo</i> samples. We performed two kinds of treatment on the cells (from an overnight culture): | ||
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<tr><td colspan=6 align=center valign=center> | <tr><td colspan=6 align=center valign=center> | ||
− | <h3>Discussion and | + | <h3>Discussion and Conclusion</h3> |
<p> | <p> | ||
We purified and proved the functionality of the Cas13a enzyme, chose Lbu for its better activity, optimized the concentrations in our detection scheme and found the detection limit to be in the range of 10 nM target RNA. We found that we could detect RNA from <i>in vivo</i> sources, with full RNA extraction, but possibly also from simply lysed cells. This makes this module (the Cas13a detection circuit) the best characterized and most promising module of our platform. It gives fast, high fluorescence signals for low target RNA concentration, and can be combined with our amplification module, which would use heat lysis (80°C) followed by reverse transcription, RPA and transcription (room temperature).</p> | We purified and proved the functionality of the Cas13a enzyme, chose Lbu for its better activity, optimized the concentrations in our detection scheme and found the detection limit to be in the range of 10 nM target RNA. We found that we could detect RNA from <i>in vivo</i> sources, with full RNA extraction, but possibly also from simply lysed cells. This makes this module (the Cas13a detection circuit) the best characterized and most promising module of our platform. It gives fast, high fluorescence signals for low target RNA concentration, and can be combined with our amplification module, which would use heat lysis (80°C) followed by reverse transcription, RPA and transcription (room temperature).</p> |
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