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<center><img src="https://static.igem.org/mediawiki/2017/e/e7/Experimap.jpg" style="width:540px;height:360px;"></center> | <center><img src="https://static.igem.org/mediawiki/2017/e/e7/Experimap.jpg" style="width:540px;height:360px;"></center> | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | + | 3 toehold switches with “good” predicted performance were chosen to target each RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). (e.g. H5-1, H5-2 and H5-3 to detect H5 RNA). The figure above shows the detection region of each toehold switch. Before constructing the toehold switches, we ensured all the switches passed our modelling criteria. | |
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− | <p><h3>Construction of toehold switch and trigger- expressing plasmid</h3></p> | + | <p><h3>Construction of toehold switch and trigger-expressing plasmid</h3></p> |
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | + | The upper picture showed the general structure of our toehold switch. 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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− | Below is a table | + | Below is a table with the information of the backbone: |
<table width="79%"> | <table width="79%"> | ||
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<td><b>Backbone</b></td> | <td><b>Backbone</b></td> | ||
<td>pSB4C5</td> | <td>pSB4C5</td> | ||
− | <td> | + | <td>pSB1K3</td> |
</tr> | </tr> | ||
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<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | + | These two backbones with different Ori and antibiotic resistance genes were used because they will be used in the following experiments. Two co-transformed plasmids should not have the same type of origin of replication (Ori), or otherwise, they will compete for the replication machinery and affect the copy number. In addition, having two different antibiotic resistance genes avoid dropping out of either one of the plasmids during selection. | |
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− | <p><h3>In vivo assay: co- transformation in E. coli</h3></p> | + | <p><h3><i>In vivo</i> assay: co-transformation in <i>E. coli</i></h3></p> |
<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 microplane 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>In vitro 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 choose the workable switch to test in a cell free system. We expressed: the toehold switch plasmid, toehold switch and trigger pair, J61002(constitutive mRFP generator; positive control) and a luciferease-expressing plasmid (positive control provided by the manufacturer) in separate cell free reactions. Negative control was done by replacing DNA with water. 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 | + | We used the Promega S30 T7 High-Yield Protein Expression System as our cell free system. After the in vivo test, we choose the workable switch to test in a cell free system. We expressed: the toehold switch plasmid, toehold switch and trigger pair, J61002(constitutive mRFP generator; positive control) and a luciferease-expressing plasmid (positive control provided by the manufacturer) in separate cell free reactions. Negative control was done by replacing DNA with water. 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 50μl, followed by incubating at 37C for an hour. The florescent signal from the reacted mixture was then determined by plate reader. Overexpression of protein in the reacted mixture was checked by SDS-PAGE. |
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Revision as of 16:42, 26 October 2017