Difference between revisions of "Part:BBa K3406007"

 
 
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__NOTOC__
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<partinfo>BBa_K3406007 short</partinfo>
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<p>LbaCas13a is a member of Cas13 protein family. It was originally found in Prevotella sp.
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MA2016 and has the common characteristic of Type VI RNA-targeting CRISPR–Cas system’s proteins, which means it could be activated by target RNA and could cleave bystander single strained RNAs. This feature could be used to target RNA and thus to detect RNA and to edit RNA.
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Besides,different Cas13 protein has different base cutting preference, and the preference of CcaCas13b is Poly A/AC
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</p>
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<partinfo>BBa_K3406007 SequenceAndFeatures</partinfo>
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===Expression and purification===
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We expressed the LbaCas13a protein in <i>E. coli</i> Rosetta2(DE3)pLysS, and purified the expressed protein by Ni-NTA purification. But because the time limitation, we didn't complete the enzyme digestion experiment before deadline.
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[[Image:The agarose gel electrophoresis of LbaCas13a purification.jpg | thumb | center | 600 px |Figure 1 The agarose gel electrophoresis of LbaCas13a purification <br>
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Lane M：Protein Marker<br>
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Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose<br>
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Lane 10：10 mM imidazole eluted protein flow through<br>
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Lane 50：50 mM imidazole eluted protein flow through<br>
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Lane 250-1 to 250-7：250 mM imidazole first to seventh eluted protein flow-through<br>
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Lane 500：500 mM imidazole eluted protein flow through<br>
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]]
  
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===Characterization===
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<h4>The cleavage activity of LbaCas13a protein</h4>
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We characterized the cleavage activity of LbaCas13a protein, and the result is shown below.
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[[Image:The cleavage activity of LbaCas13a protein.jpg | thumb | center | 1000 px |Figure 2 The cleavage activity of LbaCas13a protein
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]]
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<h5>Analysis</h5>
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As shown in Figure 2, the activity of LbaCas13a protein is very low. Meanwhile, it seems that the activity of the expressed LbaCas13a protein was independent of the presence or absence of the target sequence. We speculate that the reason is that the soluble tag was not removed. Because the MBP soluble tag is very large, and it may be in the active center of the protein, which may affect the activity of the protein. Moreover, the inaccuracy of protein concentration data may also lead to the low activity of LbaCas13a protein.

Latest revision as of 15:45, 27 October 2020

6xHis-MBP-LbaCas13a

LbaCas13a is a member of Cas13 protein family. It was originally found in Prevotella sp. MA2016 and has the common characteristic of Type VI RNA-targeting CRISPR–Cas system’s proteins, which means it could be activated by target RNA and could cleave bystander single strained RNAs. This feature could be used to target RNA and thus to detect RNA and to edit RNA. Besides,different Cas13 protein has different base cutting preference, and the preference of CcaCas13b is Poly A/AC


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 427
    Illegal BglII site found at 3417
    Illegal BglII site found at 4932
    Illegal BamHI site found at 1344
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2987
    Illegal AgeI site found at 4064
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1658
    Illegal BsaI.rc site found at 2741

Expression and purification

We expressed the LbaCas13a protein in E. coli Rosetta2(DE3)pLysS, and purified the expressed protein by Ni-NTA purification. But because the time limitation, we didn't complete the enzyme digestion experiment before deadline.

Figure 1 The agarose gel electrophoresis of LbaCas13a purification
Lane M：Protein Marker
Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose
Lane 10：10 mM imidazole eluted protein flow through
Lane 50：50 mM imidazole eluted protein flow through
Lane 250-1 to 250-7：250 mM imidazole first to seventh eluted protein flow-through
Lane 500：500 mM imidazole eluted protein flow through

Characterization

The cleavage activity of LbaCas13a protein

We characterized the cleavage activity of LbaCas13a protein, and the result is shown below.

Figure 2 The cleavage activity of LbaCas13a protein
Analysis

As shown in Figure 2, the activity of LbaCas13a protein is very low. Meanwhile, it seems that the activity of the expressed LbaCas13a protein was independent of the presence or absence of the target sequence. We speculate that the reason is that the soluble tag was not removed. Because the MBP soluble tag is very large, and it may be in the active center of the protein, which may affect the activity of the protein. Moreover, the inaccuracy of protein concentration data may also lead to the low activity of LbaCas13a protein.