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<h1 class="content-header2">Description</h1>
<section>
- <h2></h2>
- <p></p>
+ <h2 class="c-green">1. Background</h2>
+ <p>
+ Gastric cancer is the second incidence of cancer in China, and the infection rate of Helicobacter pylori (H.
+ pylori) in the high incidence of gastric cancer is more than 60%. <i>H. pylori</i> infection has been identified as a
+ major carcinogenic factor causing gastric cancer, so its detection has also been included in a common item in
+ physical examination. Early detection and effective treatment of <i>H. pylori</i> infection can effectively reduce
+ the incidence of a series of gastric diseases, such as gastric ulcers and gastric cancer. In recent years, H.
+ pylori infection has emerged as an especially big problem in China. The current infection rate of HP stands at
+ 49.6%, which is slightly higher than the global infection rate of 48.5%. Nowadays, the methods of detecting H.
+ pylori are mainly based on the C-13/c-14 test, Blood and fecal antigen testing, et.al, which is not convenient
+ for daily life.
+ </p>
+ <p>
+ Based on CRISPR pathogenic microbial detection technology was developed in recent years, and was applied to a
+ series of pathogenic microorganism detection, such as HPV and nCov2, has the advantages of being fast and
+ efficient, so the development of CRISPR rapid detection of <i>H. pylori</i> can effectively prevent or find <i>H. pylori</i>
+ infection as soon as possible, so as to reduce the incidence of gastric cancer. This CRISPR rapid detection
+ technology mainly includes two types, one is the Cas13a-based SHERLOCK system, the former for detecting
+ microorganisms with RNA as genetic material, and the Cas12a-based DETECTR system with DNA as genetic material.
+ These methods have the advantages of being fast, accurate, and easy to operate.
+ </p>
+ <p>
+ In order to develop a self-diagnostic box that could be used in daily life or even be used in under-developed
+ countries with poor medical conditions, we employed FnCas12a protein, and build up an in vitro reaction system
+ for <i>H. pylori</i> detection (Figure 1).
+ </p>
+ <div class="imager">
+ <img class="rw-100" src="https://static.igem.wiki/teams/4304/wiki/description/t-ykpao-description-01.jpg"
+ alt="">
+ <span class="figure">
+ Figure 1. the principle of our in vitro <i>H. pylori</i> detection platform
+ </span>
+ </div>
+ </section>
+
+ <section>
+ <h2 class="c-green">2. Experiment Design</h2>
+ <p>
+ Our team aims to develop an in vitro reaction system for <i>H. pylori</i> detection with FnCas12a. We synthesized
+ four DNA fragments of <i>H. pylori</i> characteristics genes as targets and used the purified FnCas12a protein to
+ recognize and cut those target genes.
+ </p>
+
+ <section>
+ <h3>General Experiment Procedure</h3>
+ <p>
+ First, we synthesized four target genes of <i>H. pylori</i>, 16S, cagA, ipaH, and invA, as our target genes of
+ FnCas12a, and the genes were synthesized into the pUC57 plasmid by a gene synthesis company. What’s more, we
+ inserted the FnCas12a gene fragment into the pET28a vector for protein expression.
+ </p>
+ <p>
+ Next, we transformed the recombinant plasmids pET28a-FnCas12a into BL21(DE3), inoculated the strain and
+ 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>.
+ </p>
+ <p>
+ Then, we obtained the sgRNAs through an in vitro transcriptional method and extracted the target sgRNAs
+ fragments. We mixed the purified FnCas12a protein, the sgRNAs, the corresponding plasmids containing DNA
+ fragments, and the reaction buffer, and we incubated the reaction system at 37°C for 2 hours, and we
+ verified the result by gel electrophoresis.
+ </p>
+ <p>
+ Finally, we designed a reporter system to easy detection the activity of FnCas12a, and then we measured the
+ fluorescence intensity.
+ </p>
+ </section>
+ </section>
+
+ <section>
+ <h2 class="c-green">3. Expected Results</h2>
+ <ol class="l-top-05">
+ <li>Successfully construct 16S, cagA, ipaH, and invA cantaining plasmids, and pET-28a-FnCas12a plasmids.</li>
+ <li>Expressed and purified FnCas12a protein.</li>
+ <li>Set up an in vitro reaction platform for FnCas12a activity detection.</li>
+ <li>Measure the fluorescence intensity of the reaction system containing the reporter system.</li>
+ </ol>
+ </section>
+
+ <section>
+ <h2 class="c-green">4. Reference</h2>
+ <ol class="l-top-05 text-justify">
+ <li>Polk, D., Peek, R. Helicobacter pylori: gastric cancer and beyond. Nat Rev Cancer 10, 403–414 (2010).
+ <a href="https://doi.org/10.1038/nrc2857">https://doi.org/10.1038/nrc2857</a></li>
+ <li>Li, M, Sun, Y, Yang, J, et al. Time trends and other sources of variation in Helicobacter pylori infection
+ in mainland China: A systematic review and meta-analysis. Helicobacter. 2020; 25:e12729.
+ <a href="https://doi.org/10.1111/hel.12729">https://doi.org/10.1111/hel.12729</a></li>
+ <li>Cover TL. Helicobacter pylori Diversity and Gastric Cancer Risk. mBio. 2016 Jan 26;7(1):e01869-15. doi:
+ 10.1128/mBio.01869-15. PMID: 26814181; PMCID: PMC4742704.</li>
+ <li>Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. CRISPR-Cas12a target binding
+ unleashes indiscriminate single-stranded DNase activity. Science. 2018 Apr 27;360(6387):436-439. doi:
+ 10.1126/science.aar6245. Epub 2018 Feb 15. Erratum in: Science. 2021 Feb 19;371(6531): PMID: 29449511;
+ PMCID: PMC6628903.</li>
+ <li>Li SY, Cheng QX, Wang JM, Li XY, Zhang ZL, Gao S, Cao RB, Zhao GP, Wang J. CRISPR-Cas12a-assisted nucleic
+ acid detection. Cell Discov. 2018 Apr 24;4:20. doi: 10.1038/s41421-018-0028-z. Erratum in: Cell Discov. 2019
+ Mar 12;5:17. PMID: 29707234; PMCID: PMC5913299.</li>
+ <li>Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados A,
+ Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan CY, Guevara H, Wadford DA, Chen JS, Chiu
+ CY. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020 Jul;38(7):870-874. doi:
+ 10.1038/s41587-020-0513-4. Epub 2020 Apr 16. PMID: 32300245; PMCID: PMC9107629.</li>
+ </ol>
</section>
</div>
</div>