Difference between revisions of "Part:BBa K3406006"
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===Characterization=== | ===Characterization=== | ||
| − | + | <h4>Collateral cleavage activity of PsmCas13b protein<h4> | |
| − | + | 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 >>Model<<to see more details. | ||
| + | [[Image:https://2020.igem.org/wiki/images/c/c5/T--ZJUT_China_B--The_Cleavage_base_preference_of_PsmCas13b_protein_upon_AC%2CAU%2CGA%2CUC.png | thumb | center | 600px |Figure 5. The Cleavage base preference of PsmCas13b protein upon AC,AU,GA,UC | ||
| + | Note: 1.The detecting system contains 4 fluorescence reporter,and AC | ||
| + | 2.The detecting parameters of the fluorescence plate reader are corresponding to the excitation and emission wavelength of the fluorophor.]] | ||
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Revision as of 14:00, 25 October 2020
6xHis/Twin strep -SUMO-PsmCas13b
waiting to be edited......
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1266
Illegal BglII site found at 3600
Illegal BamHI site found at 144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 644
Illegal NgoMIV site found at 1622 - 1000INCOMPATIBLE 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<h4>
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 >>Model<<to see more details.
File:Https://2020.igem.org/wiki/images/c/c5/T--ZJUT China B--The Cleavage base preference of PsmCas13b protein upon AC,AU,GA,UC.png Figure 5. The Cleavage base preference of PsmCas13b protein upon AC,AU,GA,UC Note: 1.The detecting system contains 4 fluorescence reporter,and AC 2.The detecting parameters of the fluorescence plate reader are corresponding to the excitation and emission wavelength of the fluorophor.
File:Https://2020.igem.org/wiki/images/c/c5/T--ZJUT China B--The Cleavage base preference of PsmCas13b protein upon AC,AU,GA,UC.png
Figure 5. The Cleavage base preference of PsmCas13b protein upon AC,AU,GA,UC Note: 1.The detecting system contains 4 fluorescence reporter,and AC 2.The detecting parameters of the fluorescence plate reader are corresponding to the excitation and emission wavelength of the fluorophor.