Difference between revisions of "Part:BBa K3406006"
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| + | ===Cloning=== | ||
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| + | 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. | ||
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| + | ===PsmCas13b protein expression and purification=== | ||
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| + | 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. | ||
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| + | [[Image:Result of Cas 13b Psm protein expression.png | thumb | left | 800 px |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. | ||
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| + | [[Image:Result of PsmCas13b purification expression.png | thumb | right | 800 px |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℃ | ||
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Revision as of 14:41, 25 October 2020
6xHis/Twin strep -SUMO-PsmCas13b
waiting to be edited......
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
waiting to be edited......
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.
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.
