Team:ZJUT China B/Engineering
Engineering
Constructed and characterized a new part：
6xHis/Twin strep -SUMO-PsmCas13b
In order to achieve our multivirus detecting goal, we need to obtain four Cas13 homologues involved in our project. Thus, we constructed biobricks and parts to express the four Cas13 homologues. All the parts we constructed can be viewed in the part registry and are all listed in our >>part page<<. For our favorite part: BBa_K3406006 , 6xHis/Twin strep -SUMO-PsmCas13b, we successfully demonstrated that it could express PsmCas13b protein as we expected. Besides, we tested the activity and base-cleavage preference of the expressed Cas13 for its characterization.
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.
1. Psm expression result：
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℃
2. Psm purification result：
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(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 >>Model<< to see more details.
Figure 4. 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 4 kind fluorescence reporters ( The information is shown in Table1 ) into the detecting system and the fluorescence results are shown in Figure 5.
Figure 5. The Cleavage base preference of PsmCas13b protein upon AC, AU, GA, UC
Note:
1. The detecting system contains four fluorescence reporters, the linking nucleotide of them are AU, AC, GA, UC respectively.
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 >>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.
For more information about constructing and characterization of this part. Please click >>BBa_K3406006 <<
Success on device
We designed a fluorescence detector for the needs of the project. This device consists of a filter turntable and a controller. It changes the wavelength on the light path through the filter wheel, so as to achieve the purpose of detecting fluorescein of a specific wavelength. Its details will be mentioned on the Detector page, please click >>Detector<<
We can prove that our device is useful. When there is only one type of fluorescein in the system, only one type of filter can transmit fluorescence. We take off the detector and shoot with the mobile phone facing the light path, as shown in the video below.
>> click << Video 1 View through the detector
We added 10uL fluorescein Cy5 ( 50uM ) to the filter paper and adjusted the exposure of the mobile phone camera to maximum, then we took this real image. In a detection cycle, only a set of filters that are used to detect Cy5 can return visible light signals, which can prove that our device is successful.
We also tested with a lower concentration of fluorescein (Cy5 1uM), and the results are shown in the following table (Table 2):
It can be clearly seen that Cy5 is at a positive level, which can prove the success of our device construction. Although we did not test other fluoresceins due to conditions, in principle, our device is totally capable of detecting the existing fluorescein one by one.
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