The upper picture showed the general structure of our toehold switch (For detailed structure, please visit our <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model"> modelling page</a>). mRFP was chosen as the reporter of our toehold switches because it is very distinguishable by naked eyes while at the same time it can be quantified by measuring the fluorescence signal using a plate reader. After having the toehold switch sequences generated by our program, we linked it with the reporter sequence and synthesized them using IDT’s sponsored gBlock synthesis service. The gBlocks were used as template and amplified by PCR. The bands with correct size were gel-purified. We inserted the purified PCR products into pSB4C5 (for switch) or pSB1K3 (for trigger) using restriction cut and ligation. Sequencing results confirmed all 30 constructs were cloned.
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The upper picture showed the general structure of our toehold switch (For detailed structure, please visit our <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model"> modelling page</a>). mRFP(E1010) was chosen as the reporter of our toehold switches because it is very distinguishable by naked eyes while at the same time it can be quantified by measuring the fluorescence signal using a plate reader. After having the toehold switch sequences generated by our program, we linked it with the reporter sequence and synthesized them using IDT’s sponsored gBlock synthesis service. The gBlocks were used as template and amplified by PCR. The bands with correct size were gel-purified. We inserted the purified PCR products into pSB4C5 (for switch) or pSB1K3 (for trigger) using restriction cut and ligation. Sequencing results confirmed all 30 constructs were cloned.
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<p> <h3>Characterization of chromoproteins</h3> </p>
<p> <h3>Characterization of chromoproteins</h3> </p>
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Different types of body fluid have different pH (5)(below figure). Since we are going to use body fluid as sample in our influenza diagnostic test, we would like to investigate if the pH in body fluid can interfere with the reporter protein we used in our test. Fluorescent signal is known to be pH-dependent because pH can change the folding and conformation of the fluorophore, and ionization states can also cause shift in the Excitation/Emission spectra (6). Therefore, we characterized the fluorescence of 2 fluorescent proteins: mRFP and amajLime at different pH. We want to find out their optimum pH and see if they are suitable to be the reporter protein in our diagnostic test.
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Different types of body fluid have different pH (5)(below figure). Since we are going to use body fluid as sample in our influenza diagnostic test, we would like to investigate if the pH in body fluid can interfere with the reporter protein we used in our test. Fluorescent signal is known to be pH-dependent because pH can change the folding and conformation of the fluorophore, and ionization states can also cause shift in the Excitation/Emission spectra (6). Therefore, we characterized the fluorescence of 2 fluorescent proteins: <a href="http://parts.igem.org/Part:BBa_E1010"> mRFP </a> and <a href="http://parts.igem.org/Part:BBa_K1033916"> amajLime</a> at different pH. We want to find out their optimum pH and see if they are suitable to be the reporter protein in our diagnostic test.