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<h6><i>First approach</i><br> | <h6><i>First approach</i><br> | ||
The complete designed construct was quite large. comprising over 4000 DNA basepairs. The construct is also made up from different protein domains, hence it was necessary to allow exchanging of the separate parts. As a result, we decided to divide our construct into 3 gBlocks, which could be connected to each other and placed in a pET28a(+) vector using Gibson Assembly. All gBlocks were synthesized by IDT.<br/><br/> | The complete designed construct was quite large. comprising over 4000 DNA basepairs. The construct is also made up from different protein domains, hence it was necessary to allow exchanging of the separate parts. As a result, we decided to divide our construct into 3 gBlocks, which could be connected to each other and placed in a pET28a(+) vector using Gibson Assembly. All gBlocks were synthesized by IDT.<br/><br/> | ||
− | + | <ul> | |
− | gBlock 1 contained a GFP sequence, followed by the first two monomers of 14-3-3.< | + | <li>gBlock 1 contained a GFP sequence, followed by the first two monomers of 14-3-3.</li> |
− | gBlock 2 contained a sequence encoding for a TEV cleavable linker, to allow protease cleavage after protein expression.< | + | <li>gBlock 2 contained a sequence encoding for a TEV cleavable linker, to allow protease cleavage after protein expression.</li> |
− | gBlock 3 contained the third and fourth monomer of 14-3-3, followed by a His tag for purification and an ExoS sequence that would allow inhibition of the last monomer.< | + | <li>gBlock 3 contained the third and fourth monomer of 14-3-3, followed by a His tag for purification and an ExoS sequence that would allow inhibition of the last monomer.</li></ul><br/> |
The Gibson assembly was attempted using NEBuilder HiFi DNA Assembly Cloning Kit, but this proved unsuccessful.<br/><br/> | The Gibson assembly was attempted using NEBuilder HiFi DNA Assembly Cloning Kit, but this proved unsuccessful.<br/><br/> |
Revision as of 13:31, 1 November 2017