Difference between revisions of "Team:TU-Eindhoven/Results/DNA"
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<h1>14-3-3 tetramer</h1> | <h1>14-3-3 tetramer</h1> | ||
| − | <h6><i>First approach</i> | + | <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. | + | 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/> |
| − | gBlock 1 contained a GFP sequence, followed by the first two monomers of 14-3-3. | + | gBlock 1 contained a GFP sequence, followed by the first two monomers of 14-3-3.<br/> |
| − | gBlock 2 contained a sequence encoding for a TEV cleavable linker, to allow protease cleavage after protein expression. | + | gBlock 2 contained a sequence encoding for a TEV cleavable linker, to allow protease cleavage after protein expression.<br/> |
| − | 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. | + | 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.<br/><br/> |
| − | The Gibson assembly was attempted using NEBuilder HiFi DNA Assembly Cloning Kit, but this proved unsuccessful. | + | The Gibson assembly was attempted using NEBuilder HiFi DNA Assembly Cloning Kit, but this proved unsuccessful.<br/><br/> |
| − | <i>Second approach</i> | + | <i>Second approach</i><br> |
We hypothesized that the unsuccessful Gibson assembly was due to the large gBlocks and large amount of inserts, so we decided to redesign our construct, by dividing it into two gBlocks: one containing the first two monomers and one containing a TEV-cleavable linker and the last two monomers. This time they did not contain ExoS or GFP groups. | We hypothesized that the unsuccessful Gibson assembly was due to the large gBlocks and large amount of inserts, so we decided to redesign our construct, by dividing it into two gBlocks: one containing the first two monomers and one containing a TEV-cleavable linker and the last two monomers. This time they did not contain ExoS or GFP groups. | ||
| − | The Gibson assembly of these constructs into the pET28a(+) vector once again proved unsuccessful. | + | The Gibson assembly of these constructs into the pET28a(+) vector once again proved unsuccessful.<br/><br/> |
| − | <i>Third approach</i> | + | <i>Third approach</i><br> |
We then decided we could use a pET28a(+) vector of our supervisor that already contained a 14-3-3 dimer. A new gBlock, comprising the third and fourth monomers and GFP, was placed behind our supervisor’s dimer using SacI and HindIII restriction sites and this finally resulted in a successful tetramer with GFP. | We then decided we could use a pET28a(+) vector of our supervisor that already contained a 14-3-3 dimer. A new gBlock, comprising the third and fourth monomers and GFP, was placed behind our supervisor’s dimer using SacI and HindIII restriction sites and this finally resulted in a successful tetramer with GFP. | ||
Gel electrophoresis after colony PCR showed that most colonies merely contained the original dimer (around 1700), but two colonies did give good results (around 4200). The sequencing services of StarSEQ confirmed that the formation of a tetrameric 14-3-3 with GFP was finally successful.</h6> | Gel electrophoresis after colony PCR showed that most colonies merely contained the original dimer (around 1700), but two colonies did give good results (around 4200). The sequencing services of StarSEQ confirmed that the formation of a tetrameric 14-3-3 with GFP was finally successful.</h6> | ||
Revision as of 13:29, 1 November 2017
