Difference between revisions of "Part:BBa K3406005"

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<partinfo>BBa_K3406005 SequenceAndFeatures</partinfo>  
 
<partinfo>BBa_K3406005 SequenceAndFeatures</partinfo>  
  
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===Cloning===
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We ordered CcaCas13b protein gene from a syntheis company. We coloned the gene and ligated it into the pET-28a(+) using One Step Colone. And then we added 6xHis/Twin strep-SUMO tag to the 5' end of the gene to construct the whole part.
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[[Image:pET-28a(+)-6xHis/Twin strep-SUMO-CcaCas13b colony PCR.jpg | thumb | center | 800 px |Figure 1 pET-28a(+)-6xHis/Twin strep-SUMO-CcaCas13b colony PCR<br>
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The result shows that XXXXXXXXX are positive colony
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]]
  
  
 
===Expression and purification===
 
===Expression and purification===
 
Because CcaCas13b protein was codon optimized, it was expressed in <i>E. coli </i>BL21(DE3). And the expressed protein was purified by Ni-NTA purification.The result is shown below.The molecular weight of CcaCas13b is 143.4 KDa which may be the banding in Lane FT and Lane 10, but the results show that from Lane 50-Lane 500, the banding appears between 41 KDa and 53 KDa. We speculate that the we inserted the 6xHis tag in the wrong site when constructing the part,thus resulting in the discrepancy between the purified protein size and the theoretical value.
 
Because CcaCas13b protein was codon optimized, it was expressed in <i>E. coli </i>BL21(DE3). And the expressed protein was purified by Ni-NTA purification.The result is shown below.The molecular weight of CcaCas13b is 143.4 KDa which may be the banding in Lane FT and Lane 10, but the results show that from Lane 50-Lane 500, the banding appears between 41 KDa and 53 KDa. We speculate that the we inserted the 6xHis tag in the wrong site when constructing the part,thus resulting in the discrepancy between the purified protein size and the theoretical value.
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[[Image:Cca purification.jpg | thumb | center | 800 px |Figure 1 The agarose gel electrophoresis of CcaCas13b purification<br>
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[[Image:Cca purification.jpg | thumb | center | 800 px |Figure 2 The agarose gel electrophoresis of CcaCas13b purification<br>
 
Lane M：Protein Marker<br>
 
Lane M：Protein Marker<br>
 
Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose<br>
 
Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose<br>

Revision as of 03:15, 27 October 2020

6xHis/Twin-strep-SUMO-CcaCas13b

CcaCas13b is a member of Cas13 protein family.It has the common characteristic of Type VI RNA-targeting CRISPR–Cas system’s proteins, which means it could be actived 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 U/UA/UC

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1436
    Illegal AgeI site found at 2288
  • 1000
    COMPATIBLE WITH RFC[1000]

Cloning

We ordered CcaCas13b protein gene from a syntheis company. We coloned the gene and ligated it into the pET-28a(+) using One Step Colone. And then we added 6xHis/Twin strep-SUMO tag to the 5' end of the gene to construct the whole part.

File:PET-28a(+)-6xHis/Twin strep-SUMO-CcaCas13b colony PCR.jpg
Figure 1 pET-28a(+)-6xHis/Twin strep-SUMO-CcaCas13b colony PCR
The result shows that XXXXXXXXX are positive colony


Expression and purification

Because CcaCas13b protein was codon optimized, it was expressed in E. coli BL21(DE3). And the expressed protein was purified by Ni-NTA purification.The result is shown below.The molecular weight of CcaCas13b is 143.4 KDa which may be the banding in Lane FT and Lane 10, but the results show that from Lane 50-Lane 500, the banding appears between 41 KDa and 53 KDa. We speculate that the we inserted the 6xHis tag in the wrong site when constructing the part,thus resulting in the discrepancy between the purified protein size and the theoretical value.

Figure 2 The agarose gel electrophoresis of CcaCas13b 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