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<center><img src="https://static.igem.org/mediawiki/2017/1/1a/CUHK_cotrans.jpg" style="width:50%;height:auto;"></center> | <center><img src="https://static.igem.org/mediawiki/2017/1/1a/CUHK_cotrans.jpg" style="width:50%;height:auto;"></center> | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | Expressing our switches in <i>E. coli</i> is a cheaper and more familiar method to validate our switches and the program. The assay is done by co-transforming switch-expressing plasmid and trigger-expressing plasmid into <i>E. coli</i> BL21 (DE3). Negative control is done by co-transforming switch-expressing plasmid and empty pSB1K3. Single colonies of the transformants were picked and grown overnight starter culture. Expression of reporter mRFP was done by shaking culture for 6 hours after 1% inoculation. Cells were harvested, washed with PBS buffer, then aliquoted in 96-well plates. Fluorescence intensity was measured by BMG ClarioStar | + | Expressing our switches in <i>E. coli</i> is a cheaper and more familiar method to validate our switches and the program. The assay is done by co-transforming switch-expressing plasmid and trigger-expressing plasmid into <i>E. coli</i> BL21 (DE3). Negative control is done by co-transforming switch-expressing plasmid and empty pSB1K3. Single colonies of the transformants were picked and grown overnight starter culture. Expression of reporter mRFP was done by shaking culture for 6 hours after 1% inoculation. Cells were harvested, washed with PBS buffer, then aliquoted in 96-well plates. Fluorescence intensity was measured by BMG ClarioStar microplate reader. We later checked for difference of florescent signal between the switch-trigger co-transformants and the negative control. In theory, if the toehold switch works as expected, the florescent signal of switch-trigger co-transformants must be higher than that of the negative control. |
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− | <p><h3>In vitro assay: Cell free system</h3> </p> | + | <p><h3><i>In vitro</i> assay: Cell free system</h3> </p> |
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | We used the Promega S30 T7 High-Yield Protein Expression System as our cell free system. After the in vivo test, we | + | We used the Promega S30 T7 High-Yield Protein Expression System as our cell free system. After the <i>in vivo</i> test, we chose the workable switches to test in cell free system. We expressed: (1) toehold switch, (2) toehold switch and trigger pair, (3) J61002(constitutive mRFP generator as a positive control of the highest possible RFP expression), (3) luciferase positive control provided by the manufacturer, and (4) negative control where DNA is replaced with water in separate cell free reactions. We followed exactly the protocol provided by the company in our experiment. The reaction was done by mixing 2 μg DNA, S30 Premix and S30 Extract together to a reaction volume of 50 μl, followed by incubating at 37˚C for an hour. The florescent signal from the reacted mixture was then determined by BMG ClarioStar plate reader. Over-expression of protein in the reaction mixture was checked by SDS-PAGE. |
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− | <p><h3>Toehold switch and trigger cloning | + | <p><h3>Toehold switch and trigger cloning tools: BBa_K2254000 & BBa_K2254001</h3></p> |
<center><img src="https://static.igem.org/mediawiki/2017/9/94/CUHK_SSD.jpg" style="width:100%;height:auto;"></center> | <center><img src="https://static.igem.org/mediawiki/2017/9/94/CUHK_SSD.jpg" 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> | <center><img src="https://static.igem.org/mediawiki/2017/7/7c/CUHK_TSD.png" style="width:100%;height:auto;"></center> | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | 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 | + | 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 oligo. 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 | + | 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. |
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Revision as of 07:00, 27 October 2017