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<center><img src="https://static.igem.org/mediawiki/2017/7/7c/CUHK_TSD.png" style="width:100%;height:auto;"></center> | <center><img src="https://static.igem.org/mediawiki/2017/7/7c/CUHK_TSD.png" style="width:100%;height:auto;"></center> | ||
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− | During our construction of switches, we realized that it would be relatively expensive to synthesis toehold switch together with the linker and reporter gene. We also want to have a convenient tool to construct and validate switches. Therefore, we constructed our toehold switch and trigger cloning tools that utilize the type IIS restriction enzyme Eco31I. Using the biobricks, user can simply construct their toehold switch or trigger by ordering 2 primer-like | + | During our construction of switches, we realized that it would be relatively expensive to synthesis toehold switch together with the linker and reporter gene. We also want to have a convenient tool to construct and validate switches. Therefore, we constructed our toehold switch and trigger cloning tools that utilize the type IIS restriction enzyme Eco31I. Using the biobricks, user can simply construct their toehold switch or trigger by ordering 2 primer-like oligos. It also utilizes screening technique that is similar to blue/white screening. User can use this biobrick to construct their toehold switches that use pT7 as the promoter and mRFP as the reporter. |
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− | To use the biobricks to clone switches and triggers, user can just order 2 oligos (similar to primer) and insert the short DNA into the biobricks by restriction cut and ligation. For the 2 oligos (about 60 nt), one oligo should contain forward toehold switch sequence with AGGG at the 5’ end, and another one should contain reverse complement toehold switch sequence with AGTA at the 5’ end. To allow convenient screening of clones, there is a constitutive promoter (J23100) and RBS (B0034) situated between two Eco31I sites. Digestion by Eco31I will remove them, and the subsequent insertion of switch will block the translation of mRFP, resulting in white colonies, whereas ligation of single digested plasmid will give red colonies. | + | To use the biobricks to clone switches and triggers, user can just order 2 oligos (similar to primer) and insert the short DNA into the biobricks by restriction cut and ligation. For the 2 oligos (about 60 nt), one oligo should contain forward toehold switch sequence with AGGG at the 5’ end, and another one should contain reverse complement toehold switch sequence with AGTA at the 5’ end. To allow convenient screening of clones, there is a constitutive promoter (J23100) and RBS (B0034) situated between the two Eco31I sites. Digestion by Eco31I will remove them, and the subsequent insertion of switch will block the translation of mRFP, resulting in white colonies, whereas ligation of single digested plasmid will give red colonies. |
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<p> <h3>Improving existing biobricks and project: Cancer switches</h3> </p> | <p> <h3>Improving existing biobricks and project: Cancer switches</h3> </p> | ||
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− | Previously, the CGU Taiwan 2015 team | + | Previously, the Chang Gung University (CGU) Taiwan 2015 team also worked on toehold switch. They designed toehold switches to detect biomarker of oral cancer. We investigated their project and found that their toehold switches have room for improvement. |
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− | Firstly, they use luciferase as reporter gene. We think that luciferase is not feasible to be used for on- site diagnosis | + | Firstly, they use luciferase as reporter gene. We think that luciferase is not feasible to be used for on-site diagnosis since the luciferase activity need to be measured by a reader. Secondly, we inputted the sequences of those oral cancer biomarkers into our program and found that a better switch can be designed by choosing another region for detection (see modelling page). Therefore, we improved their biobricks by using <i>in silico</i> design of switch and RFP as a reporter. |
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− | <p> <h3>Characterization of | + | <p> <h3>Characterization of chromoproteins</h3> </p> |
− | Different types of body fluid have different pH (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 the reporter protein we used in our test. | + | Different types of body fluid have different pH (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 (Ref). 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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Revision as of 07:21, 27 October 2017