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<h6>The DNA constructs have been designed and assembled on multiple ways, which will briefly be described here.</h6><br/><br/> | <h6>The DNA constructs have been designed and assembled on multiple ways, which will briefly be described here.</h6><br/><br/> | ||
− | < | + | <h2>14-3-3 tetramer</h2> |
<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/> | ||
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<h6><div class="Figure_2"><img src="https://static.igem.org/mediawiki/2017/8/81/T--TU-Eindhoven--CT33_in_pBAD.png"> | <h6><div class="Figure_2"><img src="https://static.igem.org/mediawiki/2017/8/81/T--TU-Eindhoven--CT33_in_pBAD.png"> | ||
− | <figcaption>Figure | + | <figcaption>Figure 2: Construct with Strep-tag, mCherry and CT33 in pBAD-DEST49</figcaption></div></h6> |
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+ | <h6>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.</h6> | ||
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Revision as of 13:39, 1 November 2017