The DNA constructs have been designed and assembled on multiple ways, which will briefly be described here.
14-3-3 tetramer
First approach
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.
gBlock 1 contained a GFP sequence, followed by the first two monomers of 14-3-3.
gBlock 2 contained a sequence encoding for a TEV cleavable linker, to allow protease cleavage after protein expression.
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.
The Gibson assembly was attempted using NEBuilder HiFi DNA Assembly Cloning Kit, but this proved unsuccessful.
Second approach
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.
Third approach
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.
Figure 1: 14-3-3 tetramer with GFP
CT33 with Strep-tag
First approach
We designed a gBlock encoding for an N-terminal Strep-tag II, followed by the mCherry fluorophore and the C-terminal CT33 sequence, in such a way that it can be placed into pBAD-DEST49 vector. This was performed using NEBuilder HiFi DNA Assembly Cloning Kit. With an expected length of approximately 1100 bp this proved successful in multiple colonies after colony PCR. This was also confirmed by sequencing.
Figure 2: Construct with Strep-tag, mCherry and CT33 in pBAD-DEST49
However, the expression of this vector and subsequent protein purification came with multiple difficulties. We hypothesized that this could be due to hairpin formation in the mRNA (Tm = 45°C according to IDT OligoAnalyzer), meaning that during translation the ATG of mCherry is recognized as start codon instead of the ATG before Strep-tag II. This way the Strep-tag II will not be expressed and protein purification is impossible. We therefore designed two sets of primers: one would insert 3 bases between the first ATG and Strep-tag II and one would insert 15 amino acids (shown in figure). This was done according to a protocol by Liu and Naismith.[1] We hoped that the first ATG would then be located outside the hairpin, so that it can be translated. Such small inserts can not be visualized on agarose gel, but sequencing confirmed that this approach was successful.
