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| | + | <h2 class="entry-title"><a href="#">Characterization</a></h2> |
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| | + | <p><span style=";font-family:宋体;font-size:19px;background:rgb(255,255,255)">1. Growth</span><span style=";font-family:Calibri;font-size:19px;background:rgb(255,255,255)"> curve of E.coli containing part K577881 in different concentration of arsenite </span><span style=";font-family:宋体;font-size:19px;background:rgb(255,255,255)">(</span><span style=";font-family:Calibri;font-size:19px;background:rgb(255,255,255)">arsenite</span><span style=";font-family:宋体;font-size:19px;background:rgb(255,255,255)">)</span><span style=";font-family:Calibri;font-size:14px"> </span></p><p><span style=";font-family:宋体;font-size:16px">Fig.</span><span style=";font-family:Calibri;font-size:16px">1</span><span style=";font-family:宋体;font-size:16px"> showed that the growth of bacteria was not affected in the range of </span><span style=";font-family:Calibri;font-size:16px">5ppb-10ppm of arsenite solution (As</span><sup><span style=";font-family:Calibri;font-size:16px;vertical-align:super">3+</span></sup><span style=";font-family:Calibri;font-size:16px">)</span><span style=";font-family:宋体;font-size:16px">. </span><span style=";font-family:Calibri;font-size:16px">However, as the concentration of arsenite increased to 50ppm, the growth of the bacteria was seriously inhibited, suggesting the toxictity to cells at this level.</span></p><p><span style=";font-family:Calibri;font-size:14px">Fig.1 </span></p><p style="text-align:center"><img alt="Characterization(图1)" width="554" height="336" src="https://2019.igem.org/wiki/images/9/9c/T--Worldshaper-Wuhan--GC-fig1.jpeg" style="max-width:100%!important;height:auto;background:url(/public/plugins/Ueditor/lang/zh-cn/images/localimage.png) no-repeat center center;border:1px solid #ddd"></p><p><span style=";font-family:Calibri;font-size:14px"> </span><br></p><p><strong><span style="font-family: Calibri;font-size: 12px">Fig.1 Growth curve of E.coli in different concentration of arsenic (As</span></strong><strong><sup><span style="font-family: Calibri;font-size: 12px;vertical-align: super">3+</span></sup></strong><strong><span style="font-family: Calibri;font-size: 12px">)</span></strong><span style=";font-family:Calibri;font-size:12px">. </span><span style=";font-family:Calibri;font-size:12px">Constructed plasmid</span><span style=";font-family:Calibri;font-size:12px"> pSB1C3-pArsR-GFP containing J33201 part</span><span style=";font-family:Calibri;font-size:12px"> was transformed into </span><span style=";font-family:Calibri;font-size:12px">E.coli DH5α</span><span style=";font-family:Calibri;font-size:12px"> strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in the fresh LB medium containing chloramphenicol 34</span><span style=";font-family:Calibri;font-size:12px">μ</span><span style=";font-family:Calibri;font-size:12px">g/ml at a ratio of 1:100, mixed well and divide into tubes. Different concentrations of arsenite(As</span><sup><span style=";font-family:Calibri;font-size:12px;vertical-align:super">3+</span></sup><span style=";font-family:Calibri;font-size:12px">) solutions were added into the test tube</span><span style=";font-family:宋体;font-size:12px">s</span><span style=";font-family:Calibri;font-size:12px">, respectively, so that the final concentration of arsenic was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm and 100ppm). Samples were taken at different time points of 0h, 1h, 2h…8h and overnight (16h) at one hour interval and </span><span style=";font-family:Calibri;font-size:12px">OD600 value were measured with a Multiskan Spectrum Microplate Reader. </span></p><p><span style=";font-family:Calibri;font-size:14px"> </span></p><p><span style=";font-family:Calibri;font-size:14px"> </span></p><p><strong><span style="font-family: 'Calibri Light';font-size: 19px;background: rgb(255, 255, 255)">2</span></strong><strong><span style="font-family: 宋体;font-size: 19px;background: rgb(255, 255, 255)">. </span></strong><strong><span style="font-family: 'Calibri Light';font-size: 19px;background: rgb(255, 255, 255)">GFP expression of E.coli under the control of J33201 induced by different concentration of arsenite (As</span></strong><strong><sup><span style="font-family: 'Calibri Light';font-size: 19px;vertical-align: super;background: rgb(255, 255, 255)">3+</span></sup></strong><strong><span style="font-family: 'Calibri Light';font-size: 19px;background: rgb(255, 255, 255)">)</span></strong></p><p><span style=";font-family:宋体;font-size:16px">As</span><span style=";font-family:Calibri;font-size:16px"> shown in Fig.2, our constructed biosensor based on J33201 part is very sensitive to arsenite(As</span><sup><span style=";font-family:Calibri;font-size:16px;vertical-align:super">3+</span></sup><span style=";font-family:Calibri;font-size:16px">) and the fluoresence signal can be detected at 50ppb arsenite. Significant dose-dependent effect was observed at the range of 50ppb-1ppm arsenite. The threshold of this biosensor is 10ppm. As the arsenite concentration increased to 50ppm, it does’t work due to the toxicity to cell at this level.</span></p><p><span style=";font-family:宋体;font-size:14px">Fig.2a</span></p><p style="text-align:center"><img alt="Characterization(图2)" width="554" height="372" src="https://2019.igem.org/wiki/images/b/b5/T--Worldshaper-wuhan--GFP1-fig2.jpeg" style="max-width:100%!important;height:auto;background:url(/public/plugins/Ueditor/lang/zh-cn/images/localimage.png) no-repeat center center;border:1px solid #ddd"></p><p><span style=";font-family:Calibri;font-size:14px"> </span><br></p><p><span style=";font-family:宋体;font-size:14px">Fig</span><span style=";font-family:Calibri;font-size:14px">.2b</span></p><p style="text-align:center"><img alt="Characterization(图3)" width="373" height="110" src="https://2019.igem.org/wiki/images/3/33/T--Worldshaper-wuhan--GFP1-fig3.jpeg" style="max-width:100%!important;height:auto;background:url(/public/plugins/Ueditor/lang/zh-cn/images/localimage.png) no-repeat center center;border:1px solid #ddd"></p><p><span style=";font-family:Calibri;font-size:14px"> </span><br></p><p><strong><span style="font-family: Calibri;font-size: 12px">Fig.2 GFP expression of E.coli under the control of J33201 in different concentration of arsenite (As</span></strong><strong><sup><span style="font-family: Calibri;font-size: 12px;vertical-align: super">3+</span></sup></strong><strong><span style="font-family: Calibri;font-size: 12px">)</span></strong><span style=";font-family:Calibri;font-size:12px">. </span><strong><span style="font-family: Calibri;font-size: 12px">(a)</span></strong><span style=";font-family:Calibri;font-size:12px"> Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of arsenite solutions were added into the test tube, respectively, so that the final concentration of arsenic was (0,5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm). Samples were taken at overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time. </span><strong><span style="font-family: Calibri;font-size: 12px">(b)</span></strong><span style=";font-family:Calibri;font-size:12px">The bacteria were centrifuged, and the pellets were was observed and photographed under Blue Light Gel Imager.</span></p><p><span style=";font-family:Calibri;font-size:14px"> </span></p><p><span style=";font-family:Calibri;font-size:16px">As it is shown in </span><span style=";font-family:宋体;font-size:16px">F</span><span style=";font-family:Calibri;font-size:16px">ig.3</span><span style=";font-family:宋体;font-size:16px">, </span><span style=";font-family:Calibri;font-size:16px">our constructed biosensor by J33201 reacted rapidly in arsenite solution. When the arsenite is added for 4 hours, the corresponding fluorescence signal can be detected at 100ppb. As time increased, weak signal can be detected at 50ppb, indicating that the biosensor can work within 4 hours. </span></p><p style="text-align:center"><img alt="Characterization(图4)" width="554" height="372" src="https://2019.igem.org/wiki/images/3/36/T--Worldshaper-wuhan--GFP2-fig4.jpeg" style="max-width:100%!important;height:auto;background:url(/public/plugins/Ueditor/lang/zh-cn/images/localimage.png) no-repeat center center;border:1px solid #ddd"></p><p><span style=";font-family:Calibri;font-size:14px"> </span><br></p><p><strong><span style="font-family: Calibri;font-size: 12px">Fig.3 GFP expression of E.coli in different concentration of arsenite (As3+) at different time points under the control of J33201.</span></strong><span style=";font-family:Calibri;font-size:12px"> Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol resistance at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6.</span><span style=";font-family:Calibri;font-size:12px"> </span><span style=";font-family:Calibri;font-size:12px">Different concentrations of arsenite solutions were added into the test tube, respectively, so that the final concentration of arsenic was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm). </span><span style=";font-family:Calibri;font-size:12px">Samples were taken at different time points of 0h, 2h, 4h, 6h and overnight (16h).</span><span style=";font-family:Calibri;font-size:12px"> GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time</span></p><p><span style=";font-family:Calibri"> </span></p><p><span style=";font-family:Calibri;font-size:16px">To summary, our biosensor constructed on the basis of J33201 is sens</span><span style=";font-family:宋体;font-size:16px">itive and fast. </span><span style=";font-family:Calibri;font-size:16px">The biosensor can detect 100ppb-10ppm arsenite (As3+) in water within 4 hours, and the minimum detection limit can reach 50ppb with the extension of time.</span></p><p><br></p> |
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