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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> | + | <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><br><br> |
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+ | <h6><div class="Figure_3"><img src="https://static.igem.org/mediawiki/2017/6/60/T--TU-Eindhoven--hairpin.png"> | ||
+ | <figcaption>Figure 3: First ATG incorporated into possible hairpin formation</figcaption></div></h6> | ||
+ | <br><br> | ||
+ | |||
+ | <h6><div class="Figure_3"><img src="https://static.igem.org/mediawiki/2017/d/dc/T--TU-Eindhoven--methionine.png"> | ||
+ | <figcaption>Figure 3: Primers insert extra 15 amino acids</figcaption></div></h6> | ||
+ | <br><br> | ||
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Revision as of 13:43, 1 November 2017