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| | <tr><td colspan=6 align=center valign=center> | | <tr><td colspan=6 align=center valign=center> |
| − | <h3>Cas13a characterization</h3> | + | <h3>Proof-of-concept</h3> |
| − | <h4>Protein cloning, expression and purification</h4>
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| | <p> | | <p> |
| − | We decided to compare 3 versions of Cas13a that were previously characterized in the literature [Gootenberg, Doudna]: 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). Lbu and Lsh were expressed in E.coli Rosetta2, as the sequences were not codon-optimized, and Lwa was expressed in E.coli BL21 (DE3) star. We created three Biobricks from the Lwa sequence: BBa_K2323000 (containing the Lwa coding sequence and a Tphi terminator), BBa_K2323001 (where a 6xHis/Twin strep tag and a SUMO tag are added to the N-terminal end of BBa_K2323000), and BBa_K2323004 (where BBa_K2323001 is preceded by the T7 promoter and the Elowitz RBS). We improved the TEV-protease BBa_K1319008 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 3 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). Lbu and Lsh were expressed in E.coli Rosetta2, as the sequences were not codon-optimized, and Lwa was expressed in E.coli 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 by tagging it with a 6xHis tag, purified it and successfully used it for the TEV cleavage of our Cas13a proteins. </p> |
| | + | <p> |
| | + | We followed the purification protocols from literature, and found that although the His-purification and the tag cleavage steps worked as expected, the cation-exchange purification step failed, and we systematically lost our proteins. We still completed the size-exclusion purification, and our proteins with some amount of contamination. Protein purification took most of the first month of our project, due to the failure of the cation-exchange chromatography, but we eventually purified functional, if not perfectly clean, proteins. |
| | + | </p> |
| | + | </td> |
| | + | </tr> |
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| | + | <tr><td colspan=6 align=center valign=center> |
| | + | <h3>Protein cloning, expression and purification</h3> |
| | + | <p> |
| | + | To prove the functionality of Cas13a, we used the 16S rRNA sequence from E.coli as a targed sequence, since the 16S rRNA 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 in vitro 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 Figure2, where the specific activity of Lbu was controlled by taking out the crRNA and Lbu, alternatively. |
| | + | </p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
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Results: Cas13a
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Proof-of-concept
We decided to compare 3 versions of Cas13a that were previously characterized in the literature1: 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). Lbu and Lsh were expressed in E.coli Rosetta2, as the sequences were not codon-optimized, and Lwa was expressed in E.coli BL21 (DE3) star. We created three Biobricks from the Lwa sequence: BBa_K2323000 (containing the Lwa coding sequence and a Tphi terminator), BBa_K2323001 (where a 6xHis/Twin strep tag and a SUMO tag are added to the N-terminal end of BBa_K2323000), and BBa_K2323004 (where BBa_K2323001 is preceded by the T7 promoter and the Elowitz RBS). We improved the TEV-protease BBa_K1319008 by tagging it with a 6xHis tag, purified it and successfully used it for the TEV cleavage of our Cas13a proteins.
We followed the purification protocols from literature, and found that although the His-purification and the tag cleavage steps worked as expected, the cation-exchange purification step failed, and we systematically lost our proteins. We still completed the size-exclusion purification, and our proteins with some amount of contamination. Protein purification took most of the first month of our project, due to the failure of the cation-exchange chromatography, but we eventually purified functional, if not perfectly clean, proteins.
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Protein cloning, expression and purification
To prove the functionality of Cas13a, we used the 16S rRNA sequence from E.coli as a targed sequence, since the 16S rRNA 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 in vitro 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 Figure2, where the specific activity of Lbu was controlled by taking out the crRNA and Lbu, alternatively.
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