Part:BBa_K5335008
SpyCatcher-Linker-Cry6Aa2-Linker-CPPs+SpyTag-6*His
SpyCatcher and SpyTag are able to connect via isopeptides, so Cry6Aa2 will load onto VLP (virus-like particles) through these two connector proteins and act upon the release of VLP. Cell Penetrating Peptides (CPPs) fuse to the C-terminal of Cry6Aa and help the Cry6Aa2 protein enter the cells of nematode worms to trigger the necrosis pathway
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1517
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal PstI site found at 1517 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1622
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1517
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1517
Illegal AgeI site found at 692 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The SpyCatcher-SpyTag system was modified from the CnaB2 domain of Streptococcus pyogenes fibronectin binding protein. SpyCatcher is an immunoglobulin-like protein with a molecular weight of about 12 kDa, and SpyTag is a short peptide composed of 13 residues that can recognize each other with high affinity and spontaneously form isopeptide bonds.Cell Penetrating Peptides (CPPs), also known as Trojan peptides, are a family of short hydrophobic peptides that can pass through the cell membrane. A variety of cellular transmembrane peptides have been found to facilitate the uptake of various molecules by cells, such as RNA/DNA, peptides, radioisotopes, liposomes, nanoparticles, and protein molecules.In our project, we fused Cry6Aa2 with SpyCatcher and CPPs, attaching it to virus-like particles with SpyTag, releasing it into the soil environment through the cleavage of engineered bacteria, and CPPs helped our insecticidal proteins enter the nematode body for killing.
Figure 1. SpyCatcher-Cry6Aa2-CPPs+SpyTag-6*His(pET28a)plasmid vector
Characterization
Colony PCR
We designed an forward primer inside the spycatcher-linker-cry6aa2-linker-cpps gene sequence and a reverse primer on spytag-6*his to serve as a primer for our colony PCR, as this may reduce the production of false positives.We chose 2× Magic Green Taq SuperMix.It contains DNA Polymerase, dNTP, and an loading buffer system.The PCR reaction consisted of 15 μL 2 × Magic Green Taq SuperMix, 1.5 μL forward primer (3.75 mM), 1.5 μL reverse primer (3.75 mM), 11 μL ddH2O and 1 μL colony.
SDS-PAGE
Figure 2. SDS-PAGE for Bacterial toal protein
M:Protein marker;Ctrl:Empty Escherichia coli BL21(DE3).Ex:Escherichia coli BL21(DE3) that has been introduced into the expression vector.
SDS-PAGE showed that the complex protein was successfully expressed.The weight of the complex protein is 68.3 kDa, which can be observed on the SDS-PAGE: the experimental group has bands at the target weight compared to the control group.There should be a pair of bands around 4 kDa that correspond to SpyTag-6*His, but it is not visible on the SDS-PAGE. We hypothesize that the protein is too small to be displayed on 12% SDS-PAGE.
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