From c63d3b4b50e4545ab27e35ac10e90281a7d70071 Mon Sep 17 00:00:00 2001
From: Devmc <dev.mcrf@qq.com>
Date: Mon, 22 Aug 2022 21:43:30 +0800
Subject: [PATCH] contribution description engineering
---
wiki/pages/collaborations.html | 193 +--------------------------------
wiki/pages/contribution.html | 193 ++++++++++++++++++++++++++++++++-
wiki/pages/description.html | 90 ++++++++++++++-
wiki/pages/engineering.html | 152 +++++++++++++++++++++++++-
4 files changed, 431 insertions(+), 197 deletions(-)
diff --git a/wiki/pages/collaborations.html b/wiki/pages/collaborations.html
index 102367b..91e8e52 100644
--- a/wiki/pages/collaborations.html
+++ b/wiki/pages/collaborations.html
@@ -12,197 +12,8 @@
<h1 class="content-header2">Collaborations</h1>
<section>
- <h2 class="c-blue">Overview</h2>
- <p>
- Different species of <i>Streptomyces</i> produce different antibiotics, so the modification of
- <i>Streptomyces</i> is of great significance for improving the production process of antibiotics. However, the
- experimental operation method of <i>Streptomyces</i> is not as mature as <i>E. coli</i> and yeast. Many
- commonly used biotechnologies do not work well with <i>Streptomyces</i>.
- </p>
- <p>
- In the past iGEM teams, there is no very detailed and complete characterization of <i>Streptomyces</i>. For
- this reason, our team provides some valuable information for future iGME teams.
- </p>
- <p>
- We submit information on the <i>Streptomyces</i> and <i>E. coli</i> shuttle plasmid-backbone. If a future team
- is committed to genetic engineering related to <i>Streptomyces</i>, the plasmid-backbone can be used.
- Moreover, we provide a set of practical solutions for knocking out <i>Streptomyces</i> genome genes. Please
- refer to the experimental methods page for details. If there is a future team dedicated to knocking out the
- gene of <i>Streptomyces</i>, they can refer to the knockout scheme we used. In addition, we have also sorted
- out 10 groups of two-component systems in <i>S. rapamycinicus</i> that affect the metabolic pathway of
- rapamycin, of which group 10 is the gene knocked out in this project. The results show that the production of
- rapamycin is greatly improved after knockout. If a future team is also committed to improving the rapamycin
- production of <i>S. rapamycinicus</i>, they can start with these two-component systems and try to knock out or
- modify them to increase the production.
- </p>
-
- <section>
- <h3>a) Submit the shuttle plasmid-backbone, BBa_K4281002, pKC1139-backbone</h3>
- <p>pKC1139-backbone is one of the most commonly used <i>Streptomyces</i> gene knock-out vectors, which can
- shuttle in <i>E. coli</i> and <i>S. rapamycinicus</i>, and it is thermosensitivity. The vector is a
- high-copy-number plasmid. When expressed in the prokaryotic system, the Apramycin+ resistance can be used to
- screen the right colony, and the strain should be cultured at 30℃, while transformed into the <i>S.
- rapamycinicus</i>, the strain should be cultured at 37℃. This plasmid backbone can be used to express
- different proteins in the future.</p>
- </section>
-
- <section>
- <h3>b) Provide a feasible plam for knocking out the genome gene of <i>Streptomyces</i>.</h3>
- <p>Take the gene M271_14685/M271_14690 knocked out by our project as an example,
- M271-14685-14690-up-M271-14685-14690-down is the upstream and downstream homologous arm of gene
- M271_14685/M271_14690 gene, and it is cloned into pKC1139 plasmid to knock out both M271_14685 and
- M271_14690 genes through recombination. Knockout strains can be successfully obtained by screening
- single-exchange strains and double-exchange strains in turn. The design of these composite parts and the
- experimental protocol are useful for future iGEMers committed to knocking out the genome gene of <i>S.
- rapamycinicus</i>.</p>
- </section>
-
- <section>
- <h3>c) Investigate and sort out 10 groups of two-component systems that affect metabolic pathways of <i>S.
- rapamycinicus</i></h3>
- <p>Histidine kinases are an ancient conserved family of enzymes that are found in bacteria, archaebacteria,
- and <i>S. rapamycinicus</i>. They are activated by a wide range of extracellular signals and transfer
- phosphate moieties to aspartates found in response regulators. It was regulated by a <i>PhoB</i> family
- transcriptional regulator (M271_14685). Our project has shown that when knocked out the histidine kinase
- M271_14690, the yield of Rapamycin increased. That means the two-component signal transduction system can be
- used to regulate in improve the yield of Rapamycin.</p>
- <p>We also identified other kinds of the two-component signal transduction system (Table 1), such as the
- M271_02725 histidine kinase was under the regulation of the LuxR family transcriptional regulator, which is
- possessing the typical LuxR-type helix–turn–helix (HTH)-DNA binding motif. In the table below, we show the
- two-component signal transduction system in the form of grouping.</p>
- </section>
-
- <section class="m-t-3">
- <div class="table-container">
- <span class="figure">Table 1. The two-component signal system in S. rapamycinicus.</span>
-
- <table class="table-head-bg-blue">
- <tbody>
- <tr>
- <th>Part Number</th>
- <th>Protein Number</th>
- <th>Function</th>
- <th>System</th>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281005">BBa_K4281005</a></td>
- <td>M271_00895</td>
- <td>LuxR family transcriptional regulator</td>
- <td rowspan="2">group 1</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281006">BBa_K4281006</a></td>
- <td>M271_00900</td>
- <td>histidine kinase</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281007">BBa_K4281007</a></td>
- <td>M271_02035</td>
- <td>histidine kinase</td>
- <td rowspan="2">group 2</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281008">BBa_K4281008</a></td>
- <td>M271_02040</td>
- <td>transcriptional regulator</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281009">BBa_K4281009</a></td>
- <td>M271_02720</td>
- <td>LuxR family transcriptional regulator</td>
- <td rowspan="2">group 3</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281010">BBa_K4281010</a></td>
- <td>M271_02725</td>
- <td>histidine kinase</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281011">BBa_K4281011</a></td>
- <td>M271_03140</td>
- <td>histidine kinase</td>
- <td rowspan="2">group 4</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281012">BBa_K4281012</a></td>
- <td>M271_03145</td>
- <td>LuxR family transcriptional regulator</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281013">BBa_K4281013</a></td>
- <td>M271_06210</td>
- <td>transcriptional regulator</td>
- <td rowspan="2">group 5</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281014">BBa_K4281014</a></td>
- <td>M271_06215</td>
- <td>histidine kinase</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281015">BBa_K4281015</a></td>
- <td>M271_06710</td>
- <td>histidine kinase</td>
- <td rowspan="2">group 6</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281016">BBa_K4281016</a></td>
- <td>M271_06715</td>
- <td>transcriptional regulator</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281017">BBa_K4281017</a></td>
- <td>M271_09110</td>
- <td>response regulator, OmpR type</td>
- <td rowspan="2">group 7</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281018">BBa_K4281018</a></td>
- <td>M271_09115</td>
- <td>histidine kinase</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281019">BBa_K4281019</a></td>
- <td>M271_09415</td>
- <td>histidine kinase</td>
- <td rowspan="2">group 8</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281020">BBa_K4281020</a></td>
- <td>M271_09420</td>
- <td>LuxR family transcriptional regulator</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281021">BBa_K4281021</a></td>
- <td>M271_14200</td>
- <td>histidine kinase</td>
- <td rowspan="2">group 9</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281022">BBa_K4281022</a></td>
- <td>M271_14205</td>
- <td>PhoB family transcriptional regulator</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281023">BBa_K4281023</a></td>
- <td>M271_14685</td>
- <td>transcriptional regulator</td>
- <td rowspan="2">group 10</td>
- </tr>
- <tr>
- <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281024">BBa_K4281024</a></td>
- <td>M271_14690</td>
- <td>histidine kinase</td>
- </tr>
- <tr>
- </tr>
- </tbody>
- </table>
-
-
- </div>
- </section>
-
+ <h2></h2>
+ <p></p>
</section>
</div>
diff --git a/wiki/pages/contribution.html b/wiki/pages/contribution.html
index 19947cd..d426fa3 100644
--- a/wiki/pages/contribution.html
+++ b/wiki/pages/contribution.html
@@ -12,8 +12,197 @@
<h1 class="content-header2">Contribution</h1>
<section>
- <h2></h2>
- <p></p>
+ <h2 class="c-blue">Overview</h2>
+ <p>
+ Different species of <i>Streptomyces</i> produce different antibiotics, so the modification of
+ <i>Streptomyces</i> is of great significance for improving the production process of antibiotics. However, the
+ experimental operation method of <i>Streptomyces</i> is not as mature as <i>E. coli</i> and yeast. Many
+ commonly used biotechnologies do not work well with <i>Streptomyces</i>.
+ </p>
+ <p>
+ In the past iGEM teams, there is no very detailed and complete characterization of <i>Streptomyces</i>. For
+ this reason, our team provides some valuable information for future iGME teams.
+ </p>
+ <p>
+ We submit information on the <i>Streptomyces</i> and <i>E. coli</i> shuttle plasmid-backbone. If a future team
+ is committed to genetic engineering related to <i>Streptomyces</i>, the plasmid-backbone can be used.
+ Moreover, we provide a set of practical solutions for knocking out <i>Streptomyces</i> genome genes. Please
+ refer to the experimental methods page for details. If there is a future team dedicated to knocking out the
+ gene of <i>Streptomyces</i>, they can refer to the knockout scheme we used. In addition, we have also sorted
+ out 10 groups of two-component systems in <i>S. rapamycinicus</i> that affect the metabolic pathway of
+ rapamycin, of which group 10 is the gene knocked out in this project. The results show that the production of
+ rapamycin is greatly improved after knockout. If a future team is also committed to improving the rapamycin
+ production of <i>S. rapamycinicus</i>, they can start with these two-component systems and try to knock out or
+ modify them to increase the production.
+ </p>
+
+ <section>
+ <h3>a) Submit the shuttle plasmid-backbone, BBa_K4281002, pKC1139-backbone</h3>
+ <p>pKC1139-backbone is one of the most commonly used <i>Streptomyces</i> gene knock-out vectors, which can
+ shuttle in <i>E. coli</i> and <i>S. rapamycinicus</i>, and it is thermosensitivity. The vector is a
+ high-copy-number plasmid. When expressed in the prokaryotic system, the Apramycin+ resistance can be used to
+ screen the right colony, and the strain should be cultured at 30℃, while transformed into the <i>S.
+ rapamycinicus</i>, the strain should be cultured at 37℃. This plasmid backbone can be used to express
+ different proteins in the future.</p>
+ </section>
+
+ <section>
+ <h3>b) Provide a feasible plam for knocking out the genome gene of <i>Streptomyces</i>.</h3>
+ <p>Take the gene M271_14685/M271_14690 knocked out by our project as an example,
+ M271-14685-14690-up-M271-14685-14690-down is the upstream and downstream homologous arm of gene
+ M271_14685/M271_14690 gene, and it is cloned into pKC1139 plasmid to knock out both M271_14685 and
+ M271_14690 genes through recombination. Knockout strains can be successfully obtained by screening
+ single-exchange strains and double-exchange strains in turn. The design of these composite parts and the
+ experimental protocol are useful for future iGEMers committed to knocking out the genome gene of <i>S.
+ rapamycinicus</i>.</p>
+ </section>
+
+ <section>
+ <h3>c) Investigate and sort out 10 groups of two-component systems that affect metabolic pathways of <i>S.
+ rapamycinicus</i></h3>
+ <p>Histidine kinases are an ancient conserved family of enzymes that are found in bacteria, archaebacteria,
+ and <i>S. rapamycinicus</i>. They are activated by a wide range of extracellular signals and transfer
+ phosphate moieties to aspartates found in response regulators. It was regulated by a <i>PhoB</i> family
+ transcriptional regulator (M271_14685). Our project has shown that when knocked out the histidine kinase
+ M271_14690, the yield of Rapamycin increased. That means the two-component signal transduction system can be
+ used to regulate in improve the yield of Rapamycin.</p>
+ <p>We also identified other kinds of the two-component signal transduction system (Table 1), such as the
+ M271_02725 histidine kinase was under the regulation of the LuxR family transcriptional regulator, which is
+ possessing the typical LuxR-type helix–turn–helix (HTH)-DNA binding motif. In the table below, we show the
+ two-component signal transduction system in the form of grouping.</p>
+ </section>
+
+ <section class="m-t-3">
+ <div class="table-container">
+ <span class="figure">Table 1. The two-component signal system in S. rapamycinicus.</span>
+
+ <table class="table-head-bg-blue">
+ <tbody>
+ <tr>
+ <th>Part Number</th>
+ <th>Protein Number</th>
+ <th>Function</th>
+ <th>System</th>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281005">BBa_K4281005</a></td>
+ <td>M271_00895</td>
+ <td>LuxR family transcriptional regulator</td>
+ <td rowspan="2">group 1</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281006">BBa_K4281006</a></td>
+ <td>M271_00900</td>
+ <td>histidine kinase</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281007">BBa_K4281007</a></td>
+ <td>M271_02035</td>
+ <td>histidine kinase</td>
+ <td rowspan="2">group 2</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281008">BBa_K4281008</a></td>
+ <td>M271_02040</td>
+ <td>transcriptional regulator</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281009">BBa_K4281009</a></td>
+ <td>M271_02720</td>
+ <td>LuxR family transcriptional regulator</td>
+ <td rowspan="2">group 3</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281010">BBa_K4281010</a></td>
+ <td>M271_02725</td>
+ <td>histidine kinase</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281011">BBa_K4281011</a></td>
+ <td>M271_03140</td>
+ <td>histidine kinase</td>
+ <td rowspan="2">group 4</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281012">BBa_K4281012</a></td>
+ <td>M271_03145</td>
+ <td>LuxR family transcriptional regulator</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281013">BBa_K4281013</a></td>
+ <td>M271_06210</td>
+ <td>transcriptional regulator</td>
+ <td rowspan="2">group 5</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281014">BBa_K4281014</a></td>
+ <td>M271_06215</td>
+ <td>histidine kinase</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281015">BBa_K4281015</a></td>
+ <td>M271_06710</td>
+ <td>histidine kinase</td>
+ <td rowspan="2">group 6</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281016">BBa_K4281016</a></td>
+ <td>M271_06715</td>
+ <td>transcriptional regulator</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281017">BBa_K4281017</a></td>
+ <td>M271_09110</td>
+ <td>response regulator, OmpR type</td>
+ <td rowspan="2">group 7</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281018">BBa_K4281018</a></td>
+ <td>M271_09115</td>
+ <td>histidine kinase</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281019">BBa_K4281019</a></td>
+ <td>M271_09415</td>
+ <td>histidine kinase</td>
+ <td rowspan="2">group 8</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281020">BBa_K4281020</a></td>
+ <td>M271_09420</td>
+ <td>LuxR family transcriptional regulator</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281021">BBa_K4281021</a></td>
+ <td>M271_14200</td>
+ <td>histidine kinase</td>
+ <td rowspan="2">group 9</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281022">BBa_K4281022</a></td>
+ <td>M271_14205</td>
+ <td>PhoB family transcriptional regulator</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281023">BBa_K4281023</a></td>
+ <td>M271_14685</td>
+ <td>transcriptional regulator</td>
+ <td rowspan="2">group 10</td>
+ </tr>
+ <tr>
+ <td><a target="_blank" href="http://parts.igem.org/Part:BBa_K4281024">BBa_K4281024</a></td>
+ <td>M271_14690</td>
+ <td>histidine kinase</td>
+ </tr>
+ <tr>
+ </tr>
+ </tbody>
+ </table>
+
+
+ </div>
+ </section>
+
</section>
</div>
diff --git a/wiki/pages/description.html b/wiki/pages/description.html
index 500ae28..8f87e58 100644
--- a/wiki/pages/description.html
+++ b/wiki/pages/description.html
@@ -12,8 +12,94 @@
<h1 class="content-header2">Description</h1>
<section>
- <h2></h2>
- <p></p>
+ <h2 class="c-blue">1. Background</h2>
+ <p>
+ Organ transplantation is the best choice for patients with organ failure. The number of kidney transplants in
+ my country ranks second in the world. About 300,000 patients need organ transplantation each year. However,
+ immune exclusion reactions will affect the long-term survival of transplant organs. The use of
+ immunosuppressive agents can prevent immune rejection so that the long-term survival of transplant organs can
+ reduce their adverse reactions to ensure the long-term high-quality life of transplant recipients. Rapamycin
+ is a widely used clinical drug for the treatment of immune rejection, which can greatly improve the survival
+ rate of transplanted organs after surgery. The traditional physical and chemical mutagenesis screening and
+ fermentation process optimization enable the fermentation level of rapamycin to be obtained.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-blue">2. Experiment design</h2>
+
+ <section>
+ <h3>2.1 Rapamycin</h3>
+ <p>
+ Rapamycin is a new type of macrolide antibiotic, and it is a compound isolated from <i>Streptomyces</i> <i>rapamycinicus</i>
+ through its antifungal activity that inhibits the growth of <i>Candida albicans</i>, <i>Cryptococcus
+ neoformans</i>, <i>Penicillium</i>, and <i>Mucosococcus</i>. Because of its complex chemical structure, it
+ is difficult to synthesize it by chemical methods, so this medicine is low productivity and it is also
+ expensive. However, there are few studies on improving the fermentation yield of rapamycin through metabolic
+ engineering. We tried to improve the fermentation yield of rapamycin by modifying the two-component signal
+ transduction system in <i>Streptomyces</i> <i>rapamycinicus</i>.
+ </p>
+ <p>
+ The two-component system is a basic control system for organisms to sense external stimuli and regulate
+ various physiological metabolism and cell behaviors (Fig.1). It consists of histidine kinases and response
+ regulatory proteins. The main type of signal transduction system is used. The two-component system is
+ important for primary and secondary metabolism, morphological differentiation, osmotic pressure, and cell
+ wall integrity of <i>Streptomyces</i> <i>rapamycinicus</i>.
+ </p>
+ <div class="imager">
+ <img class="rw-85"
+ src="https://static.igem.wiki/teams/4281/wiki/description/t-ecnuas-description-01.jpg"
+ alt="">
+ <span class="figure">Fig.1. Two-component system schematic diagram</span>
+ </div>
+ </section>
+
+ <section>
+ <h3>2.2 General experiment procedure</h3>
+ <p>
+ To construct the engineered strain, we amplified the upstream and downstream homologous arm of gene
+ M271_14685/M271_14690, cloned it into pKC1139 plasmid, and then transfer it into ET12567/pUZ8002 competent
+ cell. Screen the correct strain and co-culture with <i>Streptomyces</i> <i>rapamycinicus</i>, choose the
+ double cross-over strain and test the fermentation yield of rapamycin by HPLC.
+ </p>
+ </section>
+ </section>
+
+
+ <section>
+ <h2 class="c-blue">3. Expect results</h2>
+ <p>
+ You can see the whole process of our experiment. We constructed plasmids in <i>E. coli</i> and then
+ transferred them into <i>Streptomyces</i> <i>rapamycinicus</i> and successfully detected the yield of
+ rapamycin was improved by creating the engineered strain. Moreover, we developed a deeper understanding of
+ complex lab processes and learned lots of experimental skills, such as PCR, DNA extraction, plasmids assembly,
+ and HPLC. We hope our result is useful for future scientific research and even be applied in clinical
+ treatment.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-blue">4. Reference </h2>
+ <ul class="l-none l-start lh-lg m-b-0">
+ <li>
+ [1] Li J, Kim SG, Blenis J. Rapamycin: One drug, many effects. Cell Metab, 2014, 19(3): 373-379.
+ </li>
+ <li>
+ [2] Benjamin D, Colombi M, Moroni C, Hall MN. Rapamycin passes the torch: a new generation of mTOR
+ inhibitors. Nat Rev <br>
+ Drug Discovery, 2011, 10(11): 868-880.
+ </li>
+ <li>
+ [3] Yoo YJ, Kim H, Park SR, Yoon YJ. An overview of rapamycin: from discovery to future perspectives. J Ind
+ Microbiol <br>
+ Biotechnol, 2017, 44(4-5): 537-553.
+ </li>
+ <li>
+ [4] Park SR, Yoo YJ, Ban YH, Yoon YJ. Biosynthesis of rapamycin and its regulation: past achievements and
+ recent progress. J <br>
+ Antibiot, 2010, 63(8): 434-441
+ </li>
+ </ul>
</section>
</div>
diff --git a/wiki/pages/engineering.html b/wiki/pages/engineering.html
index 3f8dcd3..c77807c 100644
--- a/wiki/pages/engineering.html
+++ b/wiki/pages/engineering.html
@@ -12,8 +12,156 @@
<h1 class="content-header2">Engineering</h1>
<section>
- <h2></h2>
- <p></p>
+ <h2 class="c-blue">Introduction</h2>
+ <p>
+ Organ transplantation faces the problem of immune rejection while giving patients a second life. Rapamycin is
+ a new type of macrolide antibiotic, and it is also a widely used clinical drug for the treatment of immune
+ rejection, which can greatly improve the survival rate of transplanted organs after surgery. But the drug is
+ produced at a low level and is expensive.
+ </p>
+ <p>
+ In our project we optimized the metabolic regulation network of rapamycin biosynthesis by knocking out the
+ two-component system encoding gene <i>M271_14685/M271_14690</i> in <i>Streptomyces</i> <i>rapamycinicus</i>,
+ thus bringing greater value to clinical treatment and bringing good news to patients with organ
+ transplantation.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-blue">Design</h2>
+ <p>
+ In this experiment, we amplified the 1000bp upstream and downstream gene fragments of
+ <i>M271_14685/M271_14690</i>, which are two-component systems encoding genes in <i>Streptomyces</i> <i>rapamycinicus</i>,
+ ligated them into the plasmid pKC1139, and then transformed it into the host strain <i>Streptomyces</i> <i>rapamycinicus</i>.
+ Through homologous recombination, single-crossover strains and double-crossover strains were obtained, and the
+ target gene <i>M271_14685/M271_14690</i> was knocked out to finally obtain mutant strain <i>ΔM271_14685/M271_14690</i>
+ (Fig1).
+ </p>
+ <div class="imager">
+ <img class="rw-75"
+ src="https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-01.png"
+ alt="">
+ <span class="figure">Figure 1. The two-component systems working diagram</span>
+ </div>
+ </section>
+
+ <section>
+ <h2 class="c-blue">Build</h2>
+ <p>
+ A gene knockout plasmid (pKC<i>M271_14685/M271_14690</i>) was constructed (Fig. 2). The plasmid is used to
+ knock out the two-component system encoding gene <i>M271_14685/M271_14690</i> in <i>Streptomyces</i> <i>rapamycinicus</i>.
+ The construction was confirmed by DNA sequencing.
+ </p>
+ <div class="imager">
+ <img class="rw-50"
+ src="https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-02.png"
+ alt="">
+ <span class="figure">Figure 2. Schematic map of the plasmid</span>
+ </div>
+ <p>
+ We designed the program by inserting the <i>M271_14685/14690</i> upstream homology arm gene into HindIII and
+ EcoRI sites of the pKC1139 vector. In order to build our plasmids, we amplified the gene fragments from the
+ genome of <i>Streptomyces</i> <i>rapamycinicus</i> NRRL 5491 by PCR (Figure 3), double-enzyme digestion, and
+ ligase to pKC1139 carrier.
+ </p>
+ <div class="imager">
+ <img class="rw-35"
+ src="https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-03.png"
+ alt="">
+ <span class="figure">
+ Figure 3: Gel electrophoresis diagram. <br>
+ <font class="fw-normal">
+ Line 1,3,5 are <i>M271_14685/14690</i> upstream homology arm gene, 1208bp. <br>
+ Line 2,4,6 are <i>M271_14685/14690</i> downstream homology arm gene, 1144bp.
+ </font>
+ </span>
+ </div>
+ <br>
+ <p>
+ In Figure 3, a clear and single DNA band at 1kp can be seen, indicating that the upstream and downstream
+ homology arms of <i>M271_14685/M271_14690</i> were successfully amplified by PCR.
+ </p>
+ <br>
+ <div class="imager">
+ <img class="rw-50"
+ src="https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-04.png"
+ alt="">
+ <span class="figure">
+ Figure 4: Gel electrophoresis diagram. <br>
+ <font class="fw-normal">
+ Line 1,3,5: recombinant plasmid pKC-<i>M271_14685/M271_14690</i>, 10320bp. <br>
+ Line 2,4,6: recombinant plasmid pKC-<i>M271_14685/M271_14690</i> digested with EcoRI/HindIII, 7962 bp, 2358 bp.
+ </font>
+ </span>
+ </div>
+ <br>
+ <p>
+ To verify if the recombinant plasmid is correct, we did double-enzyme-digestion. It can be seen from the
+ Figure 4 that the size of the plasmid we constructed is correct, and the identification results of double
+ enzyme digestion are also correct.
+ </p>
+ <br>
+ <p>
+ We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing
+ comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully
+ constructed.
+ </p>
+ <br>
+ <p>
+ To construct the engineering strain, we firstly transferred the recombinant plasmid into ET12567/pUZ8002
+ competent cells and screened the correct strain through 3 antibodies, and cultured it in the liquid medium.
+ Then co-cultured the <i>E.coli</i> with <i>Streptomyces</i> <i>rapamycinicus</i> and screened for the
+ cross-over strain.
+ </p>
+ <p>
+ It can be seen from Figure 5 that the gene fragments of the engineered bacteria we constructed were
+ successfully exchanged compared with the negative control. That is, the engineered bacteria that knocked out
+ the <i>M271_14685/M271_14690</i> gene were successfully obtained.
+ </p>
+ <div class="imager">
+ <img class="rw-35"
+ src="https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-05.png"
+ alt="">
+ <span class="figure">
+ Figure 5. Gel electrophoresis diagram. Line 1: double cross-over strain, 326bp, correct. <br>
+ <font class="fw-normal">
+ Line 2: control strain: <i>Streptomyces</i> <i>rapamycinicus</i> NRRL 5491, 3475bp.
+ </font>
+ </span>
+ </div>
+ </section>
+
+ <section>
+ <h2 class="c-blue">Test</h2>
+ <p>
+ Co-cultured <i>Streptomyces</i> <i>rapamycinicus</i> NRRL 5491 with <i>ΔM271_14685/M271_14690</i> in the
+ fermentation medium, we collected the sample respectively at 5d,7d,9d, and 11d. We mixed 0.5 mL samples with
+ the same volume of methanol and tested the yield of rapamycin after centrifuge. To identify the correct peek
+ of rapamycin, we use the standard rapamycin as positive control. It can be seen from Figure 8 that the <i>â–³M271_14685/m271_14690</i>
+ detected more rapamycin at the same time as standard rapamycin.
+ </p>
+ <div class="imager">
+ <img class="rw-50"
+ src="https://static.igem.wiki/teams/4281/wiki/engineering/t-ecnuas-engineering-06.png"
+ alt="">
+ <span class="figure">Figure 6. â–³<i>M271_14685/m271_14690</i> and NRRL 5491 have produced rapamycin in 11 days.</span>
+ </div>
+ <br>
+ <p>
+ It can be seen from the above figure that the rapamycin produced by our knockout <i>â–³M271_14685/m271_14690</i>
+ is much higher than that produced by NRRL 5491 (Figure 6). And the amount of rapamycin produced on the ninth
+ day reached 120mg/L.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-blue">Learn</h2>
+ <p>
+ This indicates that the knockout of the target gene <i>M271_14685/M271_14690</i> is more conducive to the
+ secretion of rapamycin by <i>Streptomyces</i> <i>rapamycinicus</i>. This suggests that the subject is feasible
+ to improve metabolic pathways by knocking out the two-component system, which can be applied to factories in
+ the near future.
+ </p>
</section>
</div>
--
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