Difference between revisions of "Part:BBa K3406006"

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Furthermore,after 100 mins of detecting, the fluorescence signal gradually became stable and finally decreased.We analysed it on the model page, click [https://2020.igem.org/Team:ZJUT_China_B/Model ZJUT_China_B Model]to see more details.
 
Furthermore,after 100 mins of detecting, the fluorescence signal gradually became stable and finally decreased.We analysed it on the model page, click [https://2020.igem.org/Team:ZJUT_China_B/Model ZJUT_China_B Model]to see more details.
  
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[[https://2020.igem.org/wiki/images/0/05/T--ZJUT_China_B--The_collateral_cleavage_activity_of_PsmCas13b_protein.png | thumb | center | 600px |Figure 5. The Cleavage base preference of PsmCas13b protein upon AC,AU,GA,UC
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[[Image:https://2020.igem.org/wiki/images/0/05/T--ZJUT_China_B--The_collateral_cleavage_activity_of_PsmCas13b_protein.png | thumb | center | 800 px |The plasmid map shows pSB1C3-His-SUMO-Cas13a which was cloned by the iGEM team Munich 2017 via Golden Gate cloning. The binding sites of used primers and all other components are annotated.
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Note: 1.The detecting system contains 4 fluorescence reporter,and AC
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2.The detecting parameters of the fluorescence plate reader are corresponding to the excitation and emission wavelength of the fluorophor.]]
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3406006 parameters</partinfo>
 
<partinfo>BBa_K3406006 parameters</partinfo>
 
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Revision as of 14:24, 25 October 2020


6xHis/Twin strep -SUMO-PsmCas13b

waiting to be edited......

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1266
    Illegal BglII site found at 3600
    Illegal BamHI site found at 144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 644
    Illegal NgoMIV site found at 1622
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 880
    Illegal SapI.rc site found at 2611

Usage and Biology

waiting to be edited......


Characterization

Collateral cleavage activity of PsmCas13b protein

After we got the expressed and purified PsmCas13 protein, we needed to verify its activity to make sure it folds properly.We tested the kinetic curve of the protein to see the dynamic change of the flourescence.As shown in the figure 3,our protein shows considerable activity in cutting flourescence reporters.We can clearly see that after adding the target, the activity of the protein is greatly improved, which enables us to tell whether the target exists in the detecting system.Interestingly,as same as LwaCas13a protein,the PsmCas13b protein also exhibits "leakage activity",which is the main interference to the detecting results. Furthermore,after 100 mins of detecting, the fluorescence signal gradually became stable and finally decreased.We analysed it on the model page, click ZJUT_China_B Modelto see more details.

File:Https://2020.igem.org/wiki/images/0/05/T--ZJUT China B--The collateral cleavage activity of PsmCas13b protein.png
The plasmid map shows pSB1C3-His-SUMO-Cas13a which was cloned by the iGEM team Munich 2017 via Golden Gate cloning. The binding sites of used primers and all other components are annotated.