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− | <h1>CT33 with Strep- | + | <h1>CT33 with Strep-tag®II</h1> |
<h2>First approach</h2> | <h2>First approach</h2> | ||
− | <h6>We designed a gBlock encoding for an N-terminal Strep- | + | <h6>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.</h6><br><br> |
<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 2: Construct with Strep- | + | <figcaption>Figure 2: Construct with Strep-tag®II, mCherry and CT33 in pBAD-DEST49</figcaption></div></h6> |
<br> | <br> | ||
− | <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- | + | <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.<sup>[1]</sup> 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><br><br> |
<h6><div class="Figure_3"><img src="https://static.igem.org/mediawiki/2017/6/60/T--TU-Eindhoven--hairpin.png"> | <h6><div class="Figure_3"><img src="https://static.igem.org/mediawiki/2017/6/60/T--TU-Eindhoven--hairpin.png"> | ||
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<h2>Second approach</h2> | <h2>Second approach</h2> | ||
− | <h6>We also planned on placing the construct in a pET28a(+) vector, with which we have more experience when it comes to expression. We designed two new gBlocks: one was quite similar to the one in pBAD-DEST49, which means it starts with Strep- | + | <h6>We also planned on placing the construct in a pET28a(+) vector, with which we have more experience when it comes to expression. We designed two new gBlocks: one was quite similar to the one in pBAD-DEST49, which means it starts with Strep-tag®II, followed by mCherry, ending with CT33. The other one starts with mCherry, followed by Strep-tag®II, ending with CT33. These are called samples SM and MS respectively. These were placed into the pET28a(+) vector using NcoI and HindIII restriction sites and these also proved to be successful in gel electrophoresis of colony PCR and sequencing. The expected lengths are around 1200 bp, with sample SM being slightly shorter than MS.</h6><br> |
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<h1>BioBricking CT33</h1> | <h1>BioBricking CT33</h1> | ||
− | <h6>Including the entire prefix and suffix in the gBlocks lead to synthesis problems, which is why only the XbaI and SpeI sites were added, which should allow clonation of the gBlock into the pSB1C3 vector at the correct location. Due to similar overhang of XbaI and SpeI, the vector could also ligate to itself, rather than to our gBlock, making this process quite inefficient. Despite this problem, we did manage to successfully create a BioBrick containing the gBlock comprising mCherry, Strep- | + | <h6>Including the entire prefix and suffix in the gBlocks lead to synthesis problems, which is why only the XbaI and SpeI sites were added, which should allow clonation of the gBlock into the pSB1C3 vector at the correct location. Due to similar overhang of XbaI and SpeI, the vector could also ligate to itself, rather than to our gBlock, making this process quite inefficient. Despite this problem, we did manage to successfully create a BioBrick containing the gBlock comprising mCherry, Strep-tag®II and CT33. This was confirmed by gel electrophoresis and sequencing.<br> |
Unfortunately the 14-3-3 tetramer with GFP is much larger and due to the high inefficiency we did not manage to properly clone it into pSB1C3. An alternative approach that we tried was the sequential digestion and ligation, starting with XbaI digestion and ligation, followed by SpeI digestion and ligation. The vector however, still preferred to ligate to itself.</h6><br/> | Unfortunately the 14-3-3 tetramer with GFP is much larger and due to the high inefficiency we did not manage to properly clone it into pSB1C3. An alternative approach that we tried was the sequential digestion and ligation, starting with XbaI digestion and ligation, followed by SpeI digestion and ligation. The vector however, still preferred to ligate to itself.</h6><br/> | ||
Latest revision as of 13:50, 10 December 2017