Difference between revisions of "Team:Worldshaper-Wuhan/Results"

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<section class="show-products-category-navigation page-borders-thick canvas-style-normal header-subtitle-none banner-alignment-center blog-layout-center project-layout-left-sidebar thumbnails-on-open-page-show-all social-icon-style-round  hide-info-footer  hide-article-author  event-show-past-events event-thumbnails event-thumbnail-size-32-standard event-date-label  event-list-show-cats event-list-date event-list-time event-list-address  event-icalgcal-links  event-excerpts event-item-back-link product-list-titles-under product-list-alignment-center product-item-size-11-square  product-gallery-size-11-square product-gallery-auto-crop show-product-price show-product-item-nav product-social-sharing newsletter-style-dark hide-opentable-icons opentable-style-dark small-button-style-solid small-button-shape-square medium-button-style-solid medium-button-shape-square large-button-style-solid large-button-shape-square image-block-poster-text-alignment-center image-block-card-dynamic-font-sizing image-block-card-content-position-center image-block-card-text-alignment-left image-block-overlap-dynamic-font-sizing image-block-overlap-content-position-center image-block-overlap-text-alignment-left image-block-collage-dynamic-font-sizing image-block-collage-content-position-top image-block-collage-text-alignment-left image-block-stack-dynamic-font-sizing image-block-stack-text-alignment-left button-style-outline button-corner-style-square tweak-product-quick-view-button-style-floating tweak-product-quick-view-button-position-bottom tweak-product-quick-view-lightbox-excerpt-display-truncate tweak-product-quick-view-lightbox-show-arrows tweak-product-quick-view-lightbox-show-close-button tweak-product-quick-view-lightbox-controls-weight-light native-currency-code-usd collection-type-page collection-5990133859cc681bb449c9ba collection-layout-default homepage mobile-style-available logo-image">
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<!--页面内容开始-->
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<p> <strong>1.&nbsp;&nbsp;&nbsp;&nbsp; Construction of the plasmids</strong>
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&nbsp; We constructed five plasmids-pEGFP-miR-21-sponge-6s; pEGFP-miR-21-sponge-2s; pHAGE-pre-miR-21; pSB1C3-miR-21-sponge-2s and pSB1C3-miR-21-sponge-6s. The plasmids of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s were used to monitor the expression of miR-21 in cells. We also used pHAGE-pre-miR-21 to manipulate miR-21 expression in cells. And the plasmids of pSB1C3-miR-21-sponge-6s and pSB1C3-miR-21-sponge-2s were submitted to 2017IGEM (part number: BBa_K2514000 and BBa_K2514001).
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<p> <strong>1.1 Construction of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s plasmid</strong>
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1.1.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.
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To monitor the expression level of miR-21 in different cells, we made two plasmids of miR-21 sponges in the 3’UTR of GFP. Firstly, we designed miR-21 sponges contains six miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-6s) and miR-21 sponges contains two miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-2s) based on the sequence of hsa-miR-21 according to the previous study (Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6). Then we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes Xho I and Hind III (Fig 1 A and B).
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<div style="padding-bottom: 29.0598%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p> <em>Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.</em>
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/"  alt="Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product."  />
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/" data-image-dimensions="468x136" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ad39652deaab95256187" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/?format=500w" data-image-resolution="500w"></div>
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<p> <em>Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.</em>
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<p>1.1.2 &nbsp;&nbsp;&nbsp; Digested pEGFP-C1 vector</p>
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We know that microRNA can bind the 3 'UTR of genes to regulate the expression, so we choose EGFP to be the detector of miR-21. We digested the pEGFP-C1 vectors with restriction enzymes Xho I and Hind III (Fig 2).
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<div class="intrinsic" style="max-width:193.0px;" id="yui_3_17_2_1_1509333291918_137">
 +
 +
<div style="padding-bottom: 49.7409%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
 +
<em>Fig 2. Electrophoresis of the digested pEGFP-c1 vector.</em>
 +
</p>
 +
" id="yui_3_17_2_1_1509333291918_136">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector."  alt="Fig 2. Electrophoresis of the digested pEGFP-c1 vector."  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector." data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector." data-image-dimensions="193x96" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ad766c3194b973e71691" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 2. Electrophoresis of the digested pEGFP-c1 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector.?format=300w" data-image-resolution="300w"></div>
 +
 +
<div class="image-caption-wrapper">
 +
<div class="image-caption">
 +
<p>
 +
<em>Fig 2. Electrophoresis of the digested pEGFP-c1 vector.</em>
 +
</p>
 +
</div>
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
</div>
 +
<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_93_1509069313608_69052">
 +
<div class="sqs-block-content">
 +
<p>
 +
<!--[if gte vml 1]>
 +
<v:shape
 +
id="_x0000_i1026" type="#_x0000_t75" style='width:193pt;height:97pt'>
 +
<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image003.png"
 +
&nbsp; o:title=""/>
 +
</v:shape>
 +
<![endif]-->
 +
</p>
 +
<p>
 +
1.1.3 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragments&nbsp; to pEGFP-c1 vector
 +
</p>
 +
<p>
 +
miR-21-sponge-6s fragments and miR-21-sponge-2s fragments were ligated to pEGFP-c1 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 3 and 4)
 +
</p>
 +
</div>
 +
</div>
 +
<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_11772">
 +
<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_156">
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 +
<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_155">
 +
 +
<div class="intrinsic" style="max-width:281.0px;" id="yui_3_17_2_1_1509333291918_154">
 +
 +
<div style="padding-bottom: 76.5125%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="" id="yui_3_17_2_1_1509333291918_153">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg"  alt="3.jpg"  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg" data-image-dimensions="281x215" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae31e4966bac11616540" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="3.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg?format=300w" data-image-resolution="300w"></div>
 +
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
</div>
 +
<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_17356">
 +
<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_173">
 +
 +
<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_172">
 +
 +
<div class="intrinsic" style="max-width:318.0px;" id="yui_3_17_2_1_1509333291918_171">
 +
 +
<div style="padding-bottom: 55.9748%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>C</p>
 +
<p>
 +
<em>
 +
Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&amp;nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map.
 +
</em>
 +
</p>
 +
" id="yui_3_17_2_1_1509333291918_170">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/"  alt="C Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and  pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map."  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/" data-image-dimensions="318x178" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae499140b7ad7ea3604f" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="C Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/?format=500w" data-image-resolution="500w"></div>
 +
 +
<div class="image-caption-wrapper">
 +
<div class="image-caption">
 +
<p>C</p>
 +
<p>
 +
<em>
 +
Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map.
 +
</em>
 +
</p>
 +
</div>
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
</div>
 +
<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_21117">
 +
<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_190">
 +
 +
<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_189">
 +
 +
<div class="intrinsic" style="max-width:193.0px;" id="yui_3_17_2_1_1509333291918_188">
 +
 +
<div style="padding-bottom: 55.9585%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
 +
<em>
 +
Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&amp;nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector.
 +
</em>
 +
</p>
 +
" id="yui_3_17_2_1_1509333291918_187">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/"  alt="Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and  pEGFP- miR-21-sponge-2s (lane 3,4) vector."  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/" data-image-dimensions="193x108" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae59652deaab95257722" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/?format=300w" data-image-resolution="300w"></div>
 +
 +
<div class="image-caption-wrapper">
 +
<div class="image-caption">
 +
<p>
 +
<em>
 +
Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector.
 +
</em>
 +
</p>
 +
</div>
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
</div>
 +
<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_12051">
 +
<div class="sqs-block-content">
 +
<p>
 +
<!--[if gte vml 1]>
 +
<v:shape
 +
id="_x0000_i1027" type="#_x0000_t75" style='width:281pt;height:214pt'>
 +
<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image005.png"
 +
&nbsp; o:title=""/>
 +
</v:shape>
 +
<![endif]-->
 +
</p>
 +
<p>
 +
<!--[if gte vml 1]>
 +
<v:shape
 +
id="_x0000_i1028" type="#_x0000_t75" style='width:318pt;height:178pt'>
 +
<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image007.png"
 +
&nbsp; o:title=""/>
 +
</v:shape>
 +
<![endif]-->
 +
</p>
 +
<p>
 +
<strong>1.2 &nbsp;&nbsp;&nbsp; Construction of miR-21- precuser expression plasmid</strong>
 +
</p>
 +
<p>
 +
1.2.1 &nbsp;&nbsp;&nbsp;&nbsp; Amplification of miR-21- precuser fragment.
 +
</p>
 +
<p>
 +
<strong>
 +
<!--[if gte vml 1]>
 +
<v:shape
 +
id="_x0000_i1029" type="#_x0000_t75" style='width:193pt;height:108pt'>
 +
<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image009.png"
 +
&nbsp; o:title=""/>
 +
</v:shape>
 +
<![endif]-->
 +
</strong>
 +
To set up a model to determent the amount of miR-21 in cells, we made a construct to express miR-21.&nbsp; We designed the specific primers of human miR-21-precuser based on the sequences of miR-21 precuser (
 +
<a href="http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000077">http://www.miRbase.org/cgi-bin/miRna_entry.pl?acc=MI0000077</a>
 +
) and amplified 72bp fragment by PCR. After that, we digested the purified PCR products with restriction enzymes Sal I and Not I (Fig 5).
 +
</p>
 +
</div>
 +
</div>
 +
<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_88253">
 +
<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_207">
 +
 +
<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_206">
 +
 +
<div class="intrinsic" style="max-width:198.0px;" id="yui_3_17_2_1_1509333291918_205">
 +
 +
<div style="padding-bottom: 54.0404%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
 +
<em>
 +
Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands.
 +
</em>
 +
</p>
 +
" id="yui_3_17_2_1_1509333291918_204">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg"  alt="Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands."  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg" data-image-dimensions="198x107" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2af200d9297adf936234e" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg?format=300w" data-image-resolution="300w"></div>
 +
 +
<div class="image-caption-wrapper">
 +
<div class="image-caption">
 +
<p>
 +
<em>
 +
Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands.
 +
</em>
 +
</p>
 +
</div>
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
</div>
 +
<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_88531">
 +
<div class="sqs-block-content">
 +
<p>
 +
<strong>
 +
<!--[if gte vml 1]>
 +
<v:shape
 +
id="_x0000_i1030" type="#_x0000_t75" style='width:198pt;height:107pt'>
 +
<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image011.png"
 +
&nbsp; o:title=""/>
 +
</v:shape>
 +
<![endif]-->
 +
</strong>
 +
1.2.2 &nbsp;Digested pHAGE-puro vector
 +
</p>
 +
<p>
 +
For
 +
<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=eukaryotic">eukaryotic</a>
 +
<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=expression">expression</a>
 +
of has-miR-21 in cells, we digested the
 +
<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=eukaryotic">eukaryotic</a>
 +
<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=expression">expression</a>
 +
vector pHAGE-puro with the same restriction enzymes Sal I and Not I&nbsp; (Fig 6).
 +
</p>
 +
</div>
 +
</div>
 +
<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_105946">
 +
<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_224">
 +
 +
<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_223">
 +
 +
<div class="intrinsic" style="max-width:198.0px;" id="yui_3_17_2_1_1509333291918_222">
 +
 +
<div style="padding-bottom: 54.5455%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
 +
<em>Fig 6. Electrophoresis of the digested pHAGE-puro vector.\</em>
 +
</p>
 +
" id="yui_3_17_2_1_1509333291918_221">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg"  alt="Fig 6. Electrophoresis of the digested pHAGE-puro vector.\"  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg" data-image-dimensions="198x108" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2af4f27ef2db556c92092" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 6. Electrophoresis of the digested pHAGE-puro vector.\" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg?format=300w" data-image-resolution="300w"></div>
 +
 +
<div class="image-caption-wrapper">
 +
<div class="image-caption">
 +
<p>
 +
<em>Fig 6. Electrophoresis of the digested pHAGE-puro vector.\</em>
 +
</p>
 +
</div>
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
 +
</div>
 +
</div>
 +
<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_106224">
 +
<div class="sqs-block-content">
 +
<p>
 +
<!--[if gte vml 1]>
 +
<v:shape
 +
id="_x0000_i1031" type="#_x0000_t75" style='width:198pt;height:107pt'>
 +
<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image013.png"
 +
&nbsp; o:title=""/>
 +
</v:shape>
 +
<![endif]-->
 +
</p>
 +
<p>
 +
1.2.3 Ligation of purified miR-21-precuser fragment to pHAGE-puro vector
 +
</p>
 +
<p>
 +
To sequence the miR-21-precuser cloning we got, the purified miR-21-precuser fragment was ligated to pHAGE-puro vector. Then we selected the positive clones by PCR and sequencing (Fig 7 and 8).
 +
</p>
 +
</div>
 +
</div>
 +
<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_121180">
 +
<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_241">
 +
 +
<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_240">
 +
 +
<div class="intrinsic" style="max-width:192.0px;" id="yui_3_17_2_1_1509333291918_239">
 +
 +
<div style="padding-bottom: 55.7292%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>A</p>
 +
" id="yui_3_17_2_1_1509333291918_238">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg"  alt="A"  />
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</noscript>
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg" data-image-dimensions="192x107" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2aff29140b7ad7ea383e0" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="A" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg?format=300w" data-image-resolution="300w"></div>
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<div class="image-caption-wrapper">
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<div class="image-caption">
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<p>A</p>
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</div>
 
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<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_258">
  
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<div class="clear"></div>
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<div class="intrinsic" style="max-width:340.0px;" id="yui_3_17_2_1_1509333291918_256">
  
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<div style="padding-bottom: 20.8824%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>B</p>
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<p>
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<em>
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Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .
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</em>
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</p>
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<p>&nbsp;</p>
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" id="yui_3_17_2_1_1509333291918_255">
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<noscript>
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/"  alt="B Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .  "  />
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</noscript>
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/" data-image-dimensions="340x71" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2b02ae31d19b2c6be28b0" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="B Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) . &nbsp;" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/?format=500w" data-image-resolution="500w"></div>
  
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<h5> Project Achievements </h5>
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<div class="image-caption-wrapper">
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<div class="image-caption">
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<p>B</p>
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<p>
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<em>
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Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .
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</em>
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</p>
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<p>&nbsp;</p>
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</div>
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</div>
  
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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</div>
  
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<ul>
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</div>
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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</ul>
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<div class="intrinsic" style="max-width:182.0px;" id="yui_3_17_2_1_1509333291918_273">
  
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<h5>Inspiration</h5>
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<div style="padding-bottom: 54.3956%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
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<p>See how other teams presented their results.</p>
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<em>Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector.</em>
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<ul>
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</p>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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" id="yui_3_17_2_1_1509333291918_272">
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<noscript>
−
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/" alt="Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector."  />
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</ul>
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</noscript>
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/" data-image-dimensions="182x99" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2b0809140b7ad7ea3904a" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/?format=300w" data-image-resolution="300w"></div>
 +
 
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<div class="image-caption-wrapper">
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<div class="image-caption">
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<p>
 +
<em>Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector.</em>
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</p>
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</div>
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</div>
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</div>
  
 
</div>
 
</div>
  
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<strong>
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visibility:visible'>
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<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image017.png"
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<![endif]-->
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1.3 &nbsp;Construction of pSB-miR-21-sponge-6s and pSB-miR-21-sponge-2s plasmid
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</strong>
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<br>
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1.3.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.
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</p>
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<p>
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<![endif]-->
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</p>
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<p>
 +
In order to submit the parts to IGM2017, we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I (Fig 9 A and B).
 +
</p>
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</div>
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</div>
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<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_19010">
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<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_292">
  
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<div style="padding-bottom: 28.0142%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
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<em>
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Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.
 +
</em>
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</p>
 +
" id="yui_3_17_2_1_1509333291918_289">
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<noscript>
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg"  alt="Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product."  />
 +
</noscript>
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg" data-image-dimensions="564x158" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f4028a652dea26877dff2d" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg?format=750w" data-image-resolution="750w"></div>
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<div class="image-caption-wrapper">
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<div class="image-caption">
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<p>
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<em>
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Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.
 +
</em>
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</p>
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</div>
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<div class="sqs-block-content">
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<p>1.3.2 &nbsp;&nbsp;&nbsp;&nbsp; Digested pSB1C3 vector</p>
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<p>
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<!--[if gte vml 1]>
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&nbsp; o:title=""/>
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<![endif]-->
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</p>
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<p>
 +
We digested the pSB1C3 vector with the same restriction enzymes EcoR I and Pst I&nbsp; (Fig 10).
 +
</p>
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</div>
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</div>
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<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_30874">
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<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_309">
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<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_308">
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<div class="intrinsic" style="max-width:268.0px;" id="yui_3_17_2_1_1509333291918_307">
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<div style="padding-bottom: 56.7164%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
 +
<em>Fig 10. Electrophoresis of the digested pSB1C3 vector.</em>
 +
</p>
 +
" id="yui_3_17_2_1_1509333291918_306">
 +
<noscript>
 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg"  alt="Fig 10. Electrophoresis of the digested pSB1C3 vector."  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg" data-image-dimensions="268x152" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f402b36926700aba0d0796" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 10. Electrophoresis of the digested pSB1C3 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg?format=300w" data-image-resolution="300w"></div>
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 +
<div class="image-caption-wrapper">
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<div class="image-caption">
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<p>
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<em>Fig 10. Electrophoresis of the digested pSB1C3 vector.</em>
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</p>
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</p>
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<p>
 +
1.3.3 &nbsp;&nbsp;&nbsp;&nbsp; Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragment&nbsp; to pSB1C3 vector
 +
</p>
 +
<p>
 +
We ligated the purified miR-21-sponge-6s or miR-21-sponge-2s fragment to pSB1C3 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 11 and 12).
 +
</p>
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg" data-image-dimensions="560x165" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f402f510952624f5425864" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="11ab.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg?format=750w" data-image-resolution="750w"></div>
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg"  alt="11c.jpg"  />
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg" data-image-dimensions="696x265" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403102774d1138b78c19a" data-type="image" data-position-mode="standard" style="left: 0%; top: -0.0781863%; width: 100%; height: 100.156%; position: absolute;" alt="11c.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg?format=750w" data-image-resolution="750w"></div>
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<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_59772">
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<div style="padding-bottom: 59.4595%; overflow: hidden;" class="image-block-wrapper  has-aspect-ratio" data-description="<p>
 +
<em>
 +
Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&amp;nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector.
 +
</em>
 +
</p>
 +
" id="yui_3_17_2_1_1509333291918_357">
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<noscript>
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg"  alt="Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and  pSB1C3- miR-21-sponge-2s (lane 3, 4) vector."  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg" data-image-dimensions="259x154" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403336c31940534284984" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg?format=300w" data-image-resolution="300w"></div>
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<div class="image-caption-wrapper">
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<em>
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Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector.
 +
</em>
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<strong>1.4</strong>
 +
&nbsp;The solid plates of each plasmid constructed (Fig 13)
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<em>Fig 13 Solid plates of five plasmids</em>
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</p>
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" id="yui_3_17_2_1_1509333291918_374">
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg"  alt="Fig 13 Solid plates of five plasmids"  />
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg" data-image-dimensions="779x576" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403756c31940534284d6b" data-type="image" data-position-mode="standard" style="left: 0%; top: -0.0323577%; width: 100%; height: 100.065%; position: absolute;" alt="Fig 13 Solid plates of five plasmids" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg?format=750w" data-image-resolution="750w"></div>
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<em>Fig 13 Solid plates of five plasmids</em>
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<p>
 +
<strong>2. The effect of miR-21 sponges in cells</strong>
 +
</p>
 +
<p>
 +
&nbsp;To detect the validity of our detection device, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug /well) with pHAGE-puro (0.25ug /well, as negative control) into 293T cells in 24 well-plate, respectively. At same times, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug/well) with pHAGE-pre-miR-21 (0.25ug/well) into 293T cells. Then the GFP fluorescence was observed under fluorescence microscopy (Fig 14 A,B,C,D). The result suggested our detection device worked very well, and overexpression of miR-21 can decrease the fluorescence intensity of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s.
 +
</p>
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<p>
 +
And we also detected the fluorescence of GFP after transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 for 24 h by plate reader (SpectraMax i3), and observed that endogenous miR-21 in cells decreased the fluorescence of GFP (Fig 15). The fluorescence of GFP in cells transfect with pEGFP-miR-21-sponge-6s was lower in that of pEGFP-miR-21-sponge-2s, because pEGFP-miR-21-sponge-6s contains six miR-21 binding sites. These results suggested that sponges with six binding sites produced stronger derepressiveeffects than sponges with two binding sites.
 +
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<em>
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Fig 14. &amp;nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm .
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 +
<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg"  alt="Fig 14.  The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm ."  />
 +
</noscript>
 +
<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg" data-image-dimensions="334x333" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403bce2c483f7f6cbd90a" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 14. &nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm ." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg?format=500w" data-image-resolution="500w"></div>
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<em>
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Fig 14. &nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm .
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Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 .
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg"  alt="Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 ."  />
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</noscript>
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg" data-image-dimensions="495x283" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403fc27ef2d09a2192ecf" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 ." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg?format=500w" data-image-resolution="500w"></div>
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<em>
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Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 .
 +
</em>
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<strong>
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2.1 The effect of miR-21 sponges as a monitor to detect the expression of miR-21
 +
</strong>
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</p>
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<p>
 +
&nbsp;&nbsp;&nbsp;&nbsp; To test the effect of miR-21 sponges as a monitor to detect the expression of miR-21, 293T cells were transfected with pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s with different concentration of pHAGE-pre-miR-21. We used the plasmid of pHAGE-pre-miR-21 to quantify miR-21 expression and calculate the copy numbers of miR-21 by using the formula listed below.
 +
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<p>
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<strong>
 +
<em>
 +
&nbsp;&nbsp;&nbsp;&nbsp; copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)
 +
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</strong>
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</p>
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<p>
 +
The standard curve of miR-21-sponge-6s and miR-21-sponge-2s were made by EXCEL, respectively (Fig 16 and 17). We find that when miR-21 copies changed the slope of the standard curve of miR-21-sponge-6s was greater than miR-21-sponge-2s which indicated miR-21-sponge-6s is more sensitive than miR-21-sponge-2s. The correlation coefficient (R2 value) of miR-21-sponge-6s was 0.9993 and R2 value of miR-21-sponge-6s was 0.8995.
 +
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<em>
 +
Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21.
 +
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4046941920221548bfb63/1509164141807/16.jpg"  alt="Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21."  />
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</noscript>
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4046941920221548bfb63/1509164141807/16.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4046941920221548bfb63/1509164141807/16.jpg" data-image-dimensions="502x256" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f4046941920221548bfb63" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4046941920221548bfb63/1509164141807/16.jpg?format=750w" data-image-resolution="750w"></div>
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Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21.
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Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s.
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<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg"  alt="Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s."  />
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<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg" data-image-dimensions="496x208" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f404aeec212d295ba9cd98" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg?format=500w" data-image-resolution="500w"></div>
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Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s.
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<strong>Conclusions and perspective</strong>
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<strong>s</strong>
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The results reported here demonstrate that miRNA sponge may be a moniter to detect the expression of miRNA in cells. And we also suggested the slope and the correlation coefficient of miR-21-sponge-6s is better than miR-21-sponge-2s. Our results for the first time to evaluate the possibility of miRNA sponge for detecting miRNA and provide a new method for diagnosis miRNA in the future. However, there are still lots of issues relative to this new method that we need to consider. Firstly, we should evaluate the sensitivity and specificity of this technique by compare with other method including real-time PCR. Next, whether miRNA sponge contained six binding sites is the best monitor should be considered.&nbsp; More strategies should be thought to improve the sensitivity and specificity of this technique by synthetic biology.
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Revision as of 03:17, 30 October 2017