Part:BBa_K3406006
6xHis/Twin strep -SUMO-PsmCas13b
Sequence and Features
- 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
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.
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 PsmCas13b protein expression
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
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
Methodology
Based on our multivirus detecting goal, we designed four kind fluorescent reporters (Table 1). It has a fluorophore on one end and a quencher on the other, The linking RNA nucleotide are AC, AU, GA, and UC respectively. And the TA and GC are DNA nucleotide which could prevent the influence of nonspecific cleavage on the detection results ( since Cas13 protein could cleave RNA nucleotide only). The fluorophores are FAM, Cy5, Texas-Red-X and VIC respectively and the excitation and emission wavelength are optimized according to the filters to combine with our detecting device. This enables us detecting the corresponding extention of the cleavage.
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.
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.
Analysis
From the result, we found that the PsmCas13b protein shows the cleavage upon AC, AU and GA while it only shows tiny cleavage activity upon UC. Interestingly, the cleavage activity of the PsmCas13b upon AC, AU and GA are different. As for GA, Psm exhibits target dependent cleavage activity. That's to say, only when the target is added, will the cleavage activity of protein be activated ( Assume that leakage cutting is not considered ) . As for AU and AC, the PsmCas13b protein shows no obvious difference between systems with target and without target. We defined this as cleavage base preference when characterizing LwaCas13a protein(click ZJUT Contribution to gain more information on "cleavage base preference" of LwaCas13a. ). The results suggest that both LwaCas13a and PsmCas13b have the cleavage base preference upon specific oligonucleotides, Lwa is AU while Psm is GA.
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