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| - | {{Team:USTC-China/temp}}
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| - | {{Team:USTC-China/temp/bar4}}
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| - | <html><a name="Aptamer-cheZ" id=""></a></html>
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| - | ==Aptamer-cheZ Device Construction==
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| - | <p> The cheZ gene was cloned from ''E.coli'' TOP10 strain using PCR. A cassete containing a theophylline-sensitive aptamer, and the cheZ gene was assembled using PCR and was subcloned into the SpeI and PstI sites of pSB1A2 containing the lac promoter.</p>
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| - | <p> Clone Primer:</p>
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| - | <p>Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGATGCAACCATCAATCAAACC-3'</p>
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| - | <p>Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGTCTATCCAACAAATCGT-3'</p>
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| - | <p> Assemble Primer:</p>
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| - | <p>Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGGGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACCCCGCTGCAAGACAACAAG ATGCAACCATCAATCAAACC-3'</p>
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| - | <p>Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGACTATCCAACAAATCGT-3'</p>
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| - | <p> Assemble condition: 96°C 10min, (96°C 30s, 56°C 30s, 72°C 1min)16 cycles and each cycle the annealing temperature increase 1°C, (96°C 30s, 72°C 30s, 72°C 1min)20 cycles,72°C 10min and holding at 4°C.</p>
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| - | <html><a name="Dose-Dependent" id=""></a></html>
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| - | ==Dose-Dependent Migration Ability of Aptamer-cheZ Device==
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| - | <p> To test the migration ability, selective media (tryptone broth with different ratio of agar(0.25%, 0.3%, 0.4%), 50μg/mL ampicllin, and various concentrations of theophylline(0mM, 0.25mM, 0.5mM, 0.5mM, 0.75mM, 1mM)) was prepared in Petri dishes(85mm dia). Diluted cell suspensions from mid-log-phase cultures were applied to the center of the plates, which were dried at room temperature at for 15 min, and incubated at 37°C for 10h, and the migration radii were determined by measuring the diameter of the outermost ring of growth. </p>
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| - | [[File:A().png|400px|center]]<p align=center>(a)</p> [[File:A(1).png|400px|center]]<p align=center>(b)</p> [[File:A(2).png|400px|center|border]]<p align=center>(c)</p>
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| - | <p align=center>Figure1.((a)0.25%agar, (b)0.3%agar, (c)0.4%agar)</p>
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| - | <p> The results(Figure1) show that the Aptamer-cheZ Device is functional, and the 0.3%agar is the best ratio for the migration experiments.</p>
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| - | <html><a name="Toggle Switch" id=""></a></html>
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| - | ==Toggle Switch Device Testing and Modifying==
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| - | <p> First, test the function of the original Toggle Switch Device from PKU. After the transformation, the ratio between the colonies with red fluorescent and the colonies with green fluorescent is 8:25. </p>
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| - | <p> Then, modify the prototype of the Toggle Switch Device from PKU. The cI gene was subcloned into the SpeI and PstI sites of pSB3t5, a low copy plasmid, containing the LuxPR promoter to create LuxPR-cI Device. After the transformation, prepare the ''E.coli'' RP1616 competence containing the LuxPR-cI Device, then use the original Toggle Switch Device to transform this competence.</p>
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| - | <p> After the transformation, the ratio between the colonies with red fluorescent and the colonies with green fluorescent is 6:1.</p>
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| - | <html><a name="Semi-Solid" id=""></a></html>
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| - | ==Semi-Solid Media with the Gadient of Theophylline==
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| - | <p> Media was prepared in 100mm square Petri dishes. Layers(15ml) of selective 0.3% agar containing theophylline(1mM, 0.25mM, 0mM) were poured in the pattern shown in Figure 2.</p>
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| - | [[File:B().jpg|center|700px]]<br/>[[File:B(1).jpg|center|700px]]<p align=center>Figure2.</p>
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| - | <p> Each layer would solidify for 50 min before applying the following layer. After all layers were applied, the media should equilibrate at room temperature for 3.0 h.</p>
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| - | <html><a name="Toggle" id=""></a></html>
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| - | ==Toggle Switch-Aptamer-cheZ Device Construction and Testing==
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| - | <p> The Aptamer-cheZ Device(without lac promoter) was subcloned into the SpeI and PstI sites of the Toggle Switch Device([http://partsregistry.org/Part:BBa_K228003 BBa_K228003]) from PKU to create the Toggle Switch -Aptamer-cheZ Device. After the transformation, diluted cell suspensions from mid-log-phase cultures were applied to the center of the Semi-Solid Media(as Figure3 shown). The plates were dried the room temperature at for 15 min, and incubated at 37°C for 10h, and the migration radii were determined by measuring the diameter of the outermost ring of growth.</p>
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| - | [[File:D().jpg|center|350px| Figure3.]]
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| - | <p align=center>Figure3.</p>
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| - | <html><a name="Toggle Switch-Aptamer-cheZ" id=""></a></html>
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| - | ==Toggle Switch-Aptamer-cheZ Device(modified) Construction and Testing==
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| - | <p> Using Toggle Switch-Aptamer-cheZ Device to transform the ''E.coli'' RP1616 competence containing the LuxPR-cI Device. After transformation, diluted cell suspensions from mid-log-phaase cultures were applied to the center of the Semi-Solid Media(as Figure3. shown). The plates were dried the room temperature at for 15 min, and incubated at 37°℃for 10h, and the migration radii were determined by measuring the diameter of the outermost ring of growth.</p>
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| - | <html><a name="Artificial" id=""></a></html>
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| - | ==Artificial Innate Immunity System Construction and Testing==
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| - | <p> For the simulation, a cassete containing a ColE7-ImmE7 complex gene([http://partsregistry.org/Part:BBa_K117001 BBa_K117001] ), Toggle Switch-Aptamer-cheZ Device, and Lysis gene([http://partsregistry.org/Part:BBa_K117000 BBa_K117000]) was assembled to create a destruction module. Then using this module to transform the ''E.coli'' RP1616 competence containing the LuxPR-cI Device. After transformation, diluted engineering bacteria and target bacteria suspensions from mid-log-phaase cultures were applied to the sites(as Figure4. shown) of the Semi-Solid Media. The plates were dried the room temperature at for 15 min, and incubated at 37°C for 10h to observe the form change of two colonies.</p>
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| - | [[File:C().jpg|center|350px| Figure4.(Colony1: engineering bacteria, Colony2: target bacteria)]]
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| - | <p align=center>Figure4.(Colony1: engineering bacteria, Colony2: target bacteria)</p>
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