|
|
| (4 intermediate revisions by the same user not shown) |
| Line 1: |
Line 1: |
| | __NOTOC__ | | __NOTOC__ |
| | <partinfo>BBa_K3406003 short</partinfo> | | <partinfo>BBa_K3406003 short</partinfo> |
| − | 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
| |
| | | | |
| | + | The coding sequence of LbaCas13a protein.It contains rare codons, so it needs to be expressed in <i>E. coli</i> strains which is rich in corresponding tRNAs like <i>E. coli</i> Rosetta2(DE3)pLysS. |
| | <partinfo>BBa_K3406003 SequenceAndFeatures</partinfo> | | <partinfo>BBa_K3406003 SequenceAndFeatures</partinfo> |
| − | ===Expression and purification===
| |
| − | 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.
| |
| − | [[Image:The agarose gel electrophoresis of LbaCas13a purification.jpg | thumb | center | 600 px |Figure 1 The agarose gel electrophoresis of LbaCas13a purification <br>
| |
| − | Lane M：Protein Marker<br>
| |
| − | Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose<br>
| |
| − | Lane 10：10 mM imidazole eluted protein flow through<br>
| |
| − | Lane 50：50 mM imidazole eluted protein flow through<br>
| |
| − | Lane 250-1 to 250-7：250 mM imidazole first to seventh eluted protein flow-through<br>
| |
| − | Lane 500：500 mM imidazole eluted protein flow through<br>
| |
| − | ]]
| |
| − |
| |
| − | ===Characterization===
| |
| − | <h4>The </h4>
| |
| − | We characterized the cleavage activity of LbaCas13a protein, and the result is shown below.
| |
| − | [[Image:The cleavage activity of LbaCas13a protein.jpg | thumb | center | 1000 px |Figure 2 The cleavage activity of LbaCas13a protein
| |
| − | ]]
| |
| − |
| |
| − | <h5>Analysis</h5>
| |
| − | 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.
| |
| − | We tested the limitation of LwaCas13a protein (Figure 16). As shown in Figure16, the LwaCas13a has a sensitivity of 10^- 13, which is quite sensitive.
| |
The coding sequence of LbaCas13a protein.It contains rare codons, so it needs to be expressed in E. coli strains which is rich in corresponding tRNAs like E. coli Rosetta2(DE3)pLysS.
