Difference between revisions of "Part:BBa K3406006"

Latest revision as of 11:53, 11 December 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 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(exhibit cleavage activity without target sequence)", 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
"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
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.

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.