Difference between revisions of "Part:BBa K3406006"

Revision as of 15:29, 25 October 2020

6xHis/Twin strep -SUMO-PsmCas13b

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Sequence and Features

Usage and Biology

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Cloning

We ordered PsmCas13b gene from a synthesis company, then we cloned the gene into pC0061. Additionally,we attached 6xHis/Twin strep tag and SUMO tag to 5' end of the gene. We transformed the ligation product into E.coli Rosetta2(DE3)pLysS and confirmed the cloning by colony-PCR and sequencing.

Figure1 Colony PCR result of 6xHis/Twin strep-SUMO PsmCas13b in E.coli Rosetta2(DE3)pLysS
Note: Lane 1,2,6,8,10 are the positive clone.

PsmCas13b protein expression and purification

We expressed the protein in E. coli Rosetta 2 (DE3) pLysS. Expressed overnight at 16°C in LB medium under the condition of 1 mM IPTG. And as the sequence contains 6×His tags, we purified the protein through Ni-NTA agarose.

• Result of PsmCas13b protein expression and purification
• 

  Figure1 Colony PCR result of 6xHis/Twin strep-SUMO PsmCas13b in E.coli Rosetta2(DE3)pLysS
  Note: Lane 1,2,6,8,10 are the positive clone.

• 

  Figure 2.Result of Cas 13b Psm protein expression
  Note:Lane M：Protein Marker, Lane PC：BSA(2.0ug), Lane NC：Cell lysate without induction, Lane 1：Cell lysate with induction for 16h at 15℃, Lane 2：Supernatant of cell lysate induction for 16h at 15℃, Lane 3：Pellet of cell lysate induction for 16h at 15℃

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.

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.

Cleavage base preference of PsmCas13b protein

To achieve our multivirus detecting goal,we further investigate the cleavage base preference of PsmCas13b protein. We added 4 kind fluorescence reporters (the linking nucleotides are AC,AU,GA,UC)into the detecting system and the fluorescence results are shown below.

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.