From 2e7257b0dd884fae15fb0903d95b2f853ab8dccf Mon Sep 17 00:00:00 2001
From: Devmc <dev.mcrf@qq.com>
Date: Thu, 15 Sep 2022 18:51:36 +0800
Subject: [PATCH] results
---
wiki/pages/part-collection.html | 278 +++++++++++++++++++++++++++++++-
wiki/pages/results.html | 158 +++++++++++++++++-
2 files changed, 430 insertions(+), 6 deletions(-)
diff --git a/wiki/pages/part-collection.html b/wiki/pages/part-collection.html
index 38f18e1..1ab096f 100644
--- a/wiki/pages/part-collection.html
+++ b/wiki/pages/part-collection.html
@@ -9,10 +9,280 @@
<div class="article">
<h1 class="content-header2">Part Collection</h1>
- <section>
- <h2></h2>
- <p></p>
- </section>
+ <table class="table-head-bg-blue table-pad-y m-b-4">
+ <tbody>
+ <tr class="text-center">
+ <th>Parts Code</th>
+ <th>Parts Name</th>
+ <th>Type I</th>
+ <th>Type II</th>
+ <th>Uplaod</th>
+ <th width="100">Uplaod sucess</th>
+ <th width="100">Complies with assembly standards</th>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304002">BBa_K4304002</a></td>
+ <td>Cas12a</td>
+ <td>Basic part</td>
+ <td>coding</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304003">BBa_K4304003</a></td>
+ <td>ipaH</td>
+ <td>Basic part</td>
+ <td>DNA</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304004">BBa_K4304004</a></td>
+ <td>invA</td>
+ <td>Basic part</td>
+ <td>DNA</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304005">BBa_K4304005</a></td>
+ <td>cagA</td>
+ <td>Basic part</td>
+ <td>DNA</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304006">BBa_K4304006</a></td>
+ <td>16S</td>
+ <td>Basic part</td>
+ <td>DNA</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304007">BBa_K4304007</a></td>
+ <td>Cas12a plasmid</td>
+ <td>Composite part</td>
+ <td>Plasmid</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304008">BBa_K4304008</a></td>
+ <td>ipaH plasmid</td>
+ <td>Composite part</td>
+ <td>Plasmid</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304009">BBa_K4304009</a></td>
+ <td>invA plasmid</td>
+ <td>Composite part</td>
+ <td>Plasmid</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304010">BBa_K4304010</a></td>
+ <td>cag plasmid</td>
+ <td>Composite part</td>
+ <td>Plasmid</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304011">BBa_K4304011</a></td>
+ <td>16S plasmid</td>
+ <td>Composite part</td>
+ <td>Plasmid</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304012">BBa_K4304012</a></td>
+ <td>T7-promoter-F</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304013">BBa_K4304013</a></td>
+ <td>cagA-crRNA1-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304014">BBa_K4304014</a></td>
+ <td>cagA-crRNA2-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304015">BBa_K4304015</a></td>
+ <td>16S-crRNA1-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304016">BBa_K4304016</a></td>
+ <td>16S-crRNA2-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304017">BBa_K4304017</a></td>
+ <td>ipaH-crRNA1-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304018">BBa_K4304018</a></td>
+ <td>ipaH-crRNA2-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304019">BBa_K4304019</a></td>
+ <td>invA-crRNA1-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304020">BBa_K4304020</a></td>
+ <td>invA-crRNA2-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Isabel Chang</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304021">BBa_K4304021</a></td>
+ <td>16S-oligoDNA-F</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304022">BBa_K4304022</a></td>
+ <td>16S-oligoDNA-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304023">BBa_K4304023</a></td>
+ <td>cagA-oligoDNA-F</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304024">BBa_K4304024</a></td>
+ <td>cagA-oligoDNA-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304025">BBa_K4304025</a></td>
+ <td>invA-oligoDNA-F</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304026">BBa_K4304026</a></td>
+ <td>invA-oligoDNA-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304027">BBa_K4304027</a></td>
+ <td>ipaH-oligoDNA-F</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304028">BBa_K4304028</a></td>
+ <td>ipaH-oligoDNA-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304029">BBa_K4304029</a></td>
+ <td>pET-Cas12a-R</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4304030">BBa_K4304030</a></td>
+ <td>pET-Cas12a-F</td>
+ <td>Basic part</td>
+ <td>primer</td>
+ <td>Eddy Liu-Lin</td>
+ <td>√</td>
+ <td>√</td>
+ </tr>
+ </tbody>
+ </table>
</div>
</div>
</div>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index 381bd10..d2c8236 100644
--- a/wiki/pages/results.html
+++ b/wiki/pages/results.html
@@ -10,8 +10,162 @@
<h1 class="content-header2">Results</h1>
<section>
- <h2></h2>
- <p></p>
+ <h2 class="c-green">1. Construction of plasmids</h2>
+ <p>
+ We designed 5 plasmids: the FnCas12 protein expression plasmid, 16S, cagA, ipaH, and invA expression plasmids.
+ Among them, the DNA fragments 16S and cagA are amplified from the genome of <i>Helicobacter Pylori</i>, and the gene
+ fragments ipaH and invA are amplified from <i>salmonella</i> and <i>shigella</i> genomic DNA respectively.
+ </p>
+ <p>
+ In order to construct our plasmids, we let the company synthesize the DNA fragments, FnCas12 was inserted into
+ the pET28a vector, and the fragments 16S, cagA, ipaH, and invA were inserted into the pUC57 vector. The
+ constructed plasmids were contained in <i>E. coli</i> strains, we streak inoculated them on LB solid medium plates
+ containing corresponding antibiotics, and incubate them at 37℃ overnight (Figure 1).
+ </p>
+ <div class="imager">
+ <img class="rw-65" src="https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-01.jpg" alt="">
+ <span class="figure">
+ Figure 1. incubate the plasmids containing strains. <br>
+ (A) Plasmid 1: pUC57-16S plasmid containing strain. <br>
+ (B) Plasmid 2: pUC57-cagA plasmid containing strain. <br>
+ (C) Plasmid 3: pUC57-invA plasmid containing strain. <br>
+ (D) Plasmid 4: pUC57-ipaH plasmid containing strain.
+ </span>
+ </div>
+ </section>
+
+ <section>
+ <h2 class="c-green">2. Extraction of oligo DNA in plasmid</h2>
+ <div class="imager">
+ <img class="rw-85" src="https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-02.jpg" alt="">
+ <span class="figure">
+ Figure 2. 1.5% TAE agarose gel electrophoresis to verify the construction of oligo DNA containing plasmids.
+ </span>
+ </div>
+ <p>
+ We used TAE agarose gel electrophoresis to testify the presence of oligo DNA in the plasmid by performing PCR
+ and then doing gel electrophoresis of the amplicons (Figure 2).
+ </p>
+ <p>
+ Our results show that a band of 200bp to 400bp is present in cagA, 16S, invA, and ipaH, but not in negative
+ control (NC) lanes. Because oligo DNA has a size between 200bp to 400bp, our result supports the fact that the
+ plasmids contain desired oligo DNA. The four plasmid transformations were successful.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-green">3. Expression and purification of Cas12a protein</h2>
+ <p>
+ We transformed the pET28a-FnCas12a expression plasmid into <i>E. coli</i> BL21(DE3) competent cells, and cultured at
+ 37℃ overnight (Figure 3A). we inoculated a single colony into LB (Kana+) culture medium, incubated overnight,
+ and then transferred the cultured medium into 1L fresh LB (Kana+) culture medium. We induced the expression of
+ FnCas12a with IPTG when the OD<sub>600</sub> was around 0.6-1.0, and cultured at 16℃ for 12h. Subsequently, we used
+ nickel affinity purification to purify the acquired Cas12a proteins from other proteins in <i>E. coli</i> (Figure
+ 3B).
+ </p>
+ <div class="imager">
+ <img class="rw-65" src="https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-03.jpg" alt="">
+ <span class="figure">
+ Figure 3. Expression and purification of protein FnCas12a. <br>
+ A. Incubate the plasmid pET28a-FnCas12a containing BL21(DE3). <br>
+ B. SDS-PAGE electrophoresis gel of Cas12a protein compared to nonspecific protein impurities.
+ </span>
+ </div>
+ <p>
+ Cas12a protein has a size of 130kDa. The SDS-PAGE electrophoresis result indicates that the Cas12a protein is
+ present in the solution we collected at 130kDa, and not present in the nonspecific protein impurities. Thus,
+ Cas12a proteins were expressed and purified with high quality.
+ </p>
+ <p>
+ Then, we tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA), using SpectraMax i3x
+ Multi-Mode Microplate Reader with the absorption peak at 562nm (Figure 4).
+ </p>
+ <div class="imager">
+ <img class="rw-65" src="https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-04.jpg" alt="">
+ <span class="figure">
+ Figure 4. BCA method standard linear regression line for calculation of protein concentration.
+ </span>
+ </div>
+ <div class="table-container">
+ <span class="figure">Table 1. Absorbance and calculated protein concentration of Cas12a 1 and Cas12a 2.</span>
+ <table class="rw-65 mx-auto">
+ <tbody>
+ <tr>
+ <th></th>
+ <th>Absorbance (L/(g·cm))</th>
+ <th>Protein Concentration (µg/ml)</th>
+ </tr>
+ <tr>
+ <td>Cas12a 1</td>
+ <td>0.2057</td>
+ <td>10.9358974</td>
+ </tr>
+ <tr>
+ <td>Cas12a 2</td>
+ <td>0.1775</td>
+ <td>7.32051282</td>
+ </tr>
+ </tbody>
+ </table>
+ </div>
+ <p>
+ With this BCA standard curve, we measured the concentration of two samples of Cas12a protein, they are 10.9
+ µg/mL and 7.32 µg/mL respectively. This result indicated that we obtained a sufficient concentration of Cas12a
+ protein.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-green">4. Cleavage experiment - cleavage of oligo DNA</h2>
+ <p>
+ In order to verify if FnCas12a we purified could precisely recognize and cut the target DNA sequence, we
+ developed an in vitro reaction platform. Firstly, we obtained the sgRNAs through an in vitro transcriptional
+ method and extracted the target sgRNAs fragments. Next, we mixed the purified FnCas12a protein, the sgRNAs,
+ the corresponding plasmids containing DNA fragments, and the reaction buffer together. Then we incubated the
+ reaction system at 37°C for 2 hours, and we verified the result by gel electrophoresis (Figure 5).
+ </p>
+ <div class="imager">
+ <img class="rw-65" src="https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-05.jpg" alt="">
+ <span class="figure">
+ Figure 5. 1.5% Agarose gel electrophoresis comparing before and after cleavage of oligo DNA. <br>
+ M = Marker, NC = Negative Control
+ </span>
+ </div>
+ <p>
+ After Cleavage is constituted of oligo DNA after cleavage by Cas12a protein and sgRNA. In contrast, NC
+ (negative control) contains oligo DNA before cleavage only. Compared to NC, The oligo DNA band is
+ significantly diminished after cleavage. This displays that the in vitro cutting experiment is successful.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-green">5. Variation of fluorescence intensity with the concentration of oligo DNA. </h2>
+ <p>
+ To assess if our Cas12a-based system worked well, we designed a reporter system by ligating a 6-FAM at the 5’
+ terminal of the target DNA probes. Then we measured the fluorescence intensity using SpectraMax i3x Multi-Mode
+ Microplate Reader every time the oligo DNA concentration increased, with the excitation of 485nm and emission
+ wavelength of 507nm.
+ </p>
+ <div class="imager">
+ <img class="rw-85" src="https://static.igem.wiki/teams/4304/wiki/results/t-ykpao-results-06.jpg" alt="">
+ <span class="figure">
+ Figure 6. Fluorescence intensity at different concentrations of oligo DNA in simulated bacteria. <br>
+ NC = negative control
+ </span>
+ </div>
+ <p>
+ The time between oligo DNA mixed with Cas12a protein and sgRNA system and measuring a sharp change in
+ fluorescence intensity within 10 minutes. As shown in the graph, as the concentration of oligo DNA increased
+ from 0 ng/μL to 4 ng/μL, the fluorescence intensity of the ssDNA fluorescent probes of the ipaH, invA, cagA,
+ and 16S systems also increased significantly. The fluorescence intensity of a system with higher oligo DNA
+ concentration is always higher than the fluorescence intensity of a system with lower oligo DNA concentration.
+ Also, our results showed that the fluorescence intensity of the oligo DNA represent group is similar to or
+ higher than the negative control (Figure 6).
+ </p>
+ <p>
+ Thus, the results indicate that our fluorescent detection experiment is successful. Cas12a protein and sgRNA
+ can recognize and cut the oligo DNA probes and the fluorescence emission can be detected.
+ </p>
</section>
</div>
</div>
--
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