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Revision as of 16:20, 25 October 2020

6xHis/Twin strep -SUMO-PsmCas13b

Sequence and Features

Usage and Biology

CRISPR Cas13 protein is the effector of clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune systems of microorganisms, which was discovered in 21 bacterial genomes. PsmCas13b 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 cleavage base preferences which may be a potential feature for multivirus detecting and the preference of PsmCas13b is Poly A/GA.

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
This colony PCR confirmed C1,C2,C6,C8,C10 as positive colony.

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
• 

  Figure2 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℃

• 

  Figure 3. Result of PsmCas13b purification expression
  Note：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 250-1：250 mM imidazole first eluted protein flow-through, Lane 250-2：250 mM imidazole second eluted protein flow-through, Lane 250-3：250 mM imidazole third eluted protein flow-through, Lane 250-4：250 mM imidazole fourth eluted protein flow-through, Lane 250-5：250 mM imidazole fifth eluted protein flow-through, Lane 250-6：250 mM imidazole sixth eluted protein flow-through, Lane 250-7：250 mM imidazole seventh eluted protein flow-through, Lane 500：500 mM imidazole eluted protein flow through

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.

Figure3 The collateral cleavage activity of PsmCas13b protein
Note: "Negative" and "PsmCas13b" are the groups with and without target sequences

Cleavage base preference of PsmCas13b protein

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

Figure5 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 fluorophore.