From 768c46cb1fb6cd8ebb30496c7680f2c9cf5c375d Mon Sep 17 00:00:00 2001
From: mriya225 <dev.mcrf@qq.com>
Date: Tue, 13 Sep 2022 18:55:45 +0800
Subject: [PATCH] implementation
---
static/utils.css | 11 +++
wiki/experiments/section-2.html | 24 +++---
wiki/pages/description.html | 2 +-
wiki/pages/implementation.html | 148 +++++++++++++++++++++++++++++++-
4 files changed, 171 insertions(+), 14 deletions(-)
diff --git a/static/utils.css b/static/utils.css
index 1cb486e..96fd943 100644
--- a/static/utils.css
+++ b/static/utils.css
@@ -578,6 +578,16 @@ section > video {
}
/*ul*/
+.article ul.l-top-05 li:nth-child(n+2),
+.article ol.l-top-05 li:nth-child(n+2) {
+ margin-top: .5rem;
+}
+
+.article ul.l-top-1 li:nth-child(n+2),
+.article ol.l-top-1 li:nth-child(n+2) {
+ margin-top: 1rem;
+}
+
.article ul.l-start,
.article ol.l-start {
padding-left: 0;
@@ -601,6 +611,7 @@ section > video {
.article ol,
.article ul {
line-height: 1.75rem;
+ font-size: 18px !important;
}
.article ul.ul-mark {
diff --git a/wiki/experiments/section-2.html b/wiki/experiments/section-2.html
index 600eef9..18676ec 100644
--- a/wiki/experiments/section-2.html
+++ b/wiki/experiments/section-2.html
@@ -156,7 +156,7 @@
<div class="collapse" id="collapse22">
<div class="card card-body">
- <ol class="ol-mark mark-quota-number l-start m-b-0">
+ <ol class="ol-mark mark-quota-number l-start m-b-0 l-top-05">
<li>
Make the agarose gel <br>
@@ -225,7 +225,7 @@
<div class="collapse" id="collapse23">
<div class="card card-body">
- <ol class="ol-mark mark-quota-number l-start m-b-0">
+ <ol class="ol-mark mark-quota-number l-start m-b-0 l-top-05">
<li>Cut down the gel which containing the target fragment and calculate the weight difference in the EP
tube from adding the gel by weighing the tube before and after adding the gel.
</li>
@@ -261,8 +261,10 @@
<div class="collapse" id="collapse24">
<div class="card card-body">
- Take 5mL of LB medium, add 5μL of Apramycin (working concentration 50μg/mL), add 100-200μL of pKC1139 strain
- and incubate in a shaker at 37℃, 220rpm overnight.
+ <p>
+ Take 5mL of LB medium, add 5μL of Apramycin (working concentration 50μg/mL), add 100-200μL of pKC1139 strain
+ and incubate in a shaker at 37℃, 220rpm overnight.
+ </p>
</div>
</div>
</section>
@@ -276,7 +278,7 @@
<div class="collapse" id="collapse25">
<div class="card card-body">
- <ol class="ol-mark mark-quota-number l-start m-b-0">
+ <ol class="ol-mark mark-quota-number l-start m-b-0 l-top-05">
<li>Bacterial solution was taken and centrifuged at 12000×g for 1 min, discard the supernatant.</li>
<li>Add 250 μL Buffer SP1 (store at 4℃, RNase A has been added) to suspension cell precipitation. (Add
bacterial suspension and suspend bacteria)
@@ -403,7 +405,7 @@
<div class="collapse" id="collapse28">
<div class="card card-body">
- <span>
+ <p>
A centrifuge tube containing <i>Escherichia coli</i> DH5a cells was placed on ice and mix with 10μL of
recombinant product. Put it on ice for 30 minutes, 42°C water bath for 90 seconds, then immediately put on
ice for 3 minutes. After that, 900μL of LB liquid medium was added into a centrifuge tube, then placed on
@@ -411,7 +413,7 @@
37°C shaker and incubated at 200×g for 1 hour. Then, the cells were centrifuged at 5000×g for 5minutues,
discarded the supernatant, and transferred the strain to an LB plate medium that contained Apramycin, and
cultured overnight at 37°C.
- </span>
+ </p>
</div>
</div>
</section>
@@ -580,13 +582,13 @@
<div class="collapse" id="collapse211">
<div class="card card-body">
- <ol class="ol-mark mark-quota-number-left l-start m-b-0">
+ <ol class="ol-mark mark-quota-number-left l-start m-b-0 l-top-05">
<li>Inoculation: inoculate pKC-M271_14685/M271_14690 to 5 mL LB (plus 5 μL of Apramycin).</li>
<li>Extract pKC-M271_14685/M271_14690 recombinant plasmids</li>
<li>
Conjugation transfer of recombinant plasmid into Streptomyces rapamycinicus. <br>
- <ol class="ol-mark mark-quota-number l-start m-b-0">
+ <ol class="ol-mark mark-quota-number l-start m-b-0 l-top-05">
<li>Use 1 μL of recombinant plasmid pKC-M271_14685/M271_14690 to transform <i>Escherichia coli</i>
ET12567/pUZ8002 competent cells, coat LB solid plates with chloramphenicol, kanamycin, and
Apramycin, and incubate overnight at 37°C. Inoculate colonies from the plates into 4mL LB liquid
@@ -619,7 +621,7 @@
<div class="collapse" id="collapse212">
<div class="card card-body">
- <ol class="ol-mark mark-quota-number-left l-start m-b-0">
+ <ol class="ol-mark mark-quota-number-left l-start m-b-0 l-top-05">
<li>
Screening for single cross-over strains <br>
The strains grown in the previous step were scribed on the oat solid medium containing Apramycin and
@@ -650,7 +652,7 @@
<div class="collapse" id="collapse213">
<div class="card card-body">
- <ol class="ol-mark mark-quota-number-left l-start m-b-0">
+ <ol class="ol-mark mark-quota-number-left l-start m-b-0 l-top-05">
<li>
The correct strain was streaked on the oat solid medium and cultured at 30°C for 7-9 days. A colony was
inoculated into 4 ml streptomyces seed medium and cultured at 28°C for 24 hours. Transferred 3 ml
diff --git a/wiki/pages/description.html b/wiki/pages/description.html
index 8f87e58..c13648a 100644
--- a/wiki/pages/description.html
+++ b/wiki/pages/description.html
@@ -80,7 +80,7 @@
<section>
<h2 class="c-blue">4. Reference </h2>
- <ul class="l-none l-start lh-lg m-b-0">
+ <ul class="l-none l-start lh-lg m-b-0 text-justify">
<li>
[1] Li J, Kim SG, Blenis J. Rapamycin: One drug, many effects. Cell Metab, 2014, 19(3): 373-379.
</li>
diff --git a/wiki/pages/implementation.html b/wiki/pages/implementation.html
index 459d02f..5d2f8b1 100644
--- a/wiki/pages/implementation.html
+++ b/wiki/pages/implementation.html
@@ -12,8 +12,152 @@
<h1 class="content-header2">Implementation</h1>
<section>
- <h2></h2>
- <p></p>
+ <h2 class="c-blue">1. Background Research</h2>
+ <p>
+ Organ transplantation is a serious domestic social issue that is widely concerned by the public due to the low
+ complementation rate. Organ donation must be evaluated by a hospital, witnessed by a third party, and signed
+ by family members. Although the number of organ donors is increasing, a considerable amount of organ donations
+ cannot meet the required clinical conditions, making organ transplantation difficult to be implemented.
+ </p>
+ <div class="imager">
+ <img class="rw-65"
+ src="https://static.igem.wiki/teams/4281/wiki/implementation/t-ecnuas-implementation-01.jpg" alt="">
+ <span class="figure">
+ <i>Number of organ donations from 2015 to 2020 in China<sup>[1]</sup></i>
+ </span>
+ </div>
+ <p>
+ Another reason is that the cost of organ transplants is too high for patients with the average income level to
+ afford. In 2016, it is reported that the cost of kidney transplantation can reach 300,000 yuan and heart
+ transplantation costs 600,000 yuan, while the average disposal income in terms of per capita income at that
+ time was merely 23, 821 yuan<sup>[2]</sup><sup>[3]</sup>. Immunosuppressive drugs alone, which can prevent the
+ activity of the
+ immune system, can cost 30,000 to 50,00 yuan per year<sup>[4]</sup>.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-blue">2. Goals and Product</h2>
+ <p>
+ Therefore, our group proposes to optimize the metabolic regulatory network of Rapamycin, the main ingredient
+ of anti-rejection drugs, and improve the fermentation level of rapamycin to improve drug manufacturing
+ efficiency. This helps to achieve our short-term goal of significantly reducing the cost of Rapamune (also
+ known as Sirolimus, an immunosuppressant widely used in kidney transplants) and increasing its productivity.
+ In the future, we hope that our product can promote the fair distribution of public medical and health
+ resources for organ transplantation and make it more affordable for patients in provinces with more
+ impoverished populations.
+ </p>
+ <p>
+ To achieve the goals we mentioned above, we designed the experiment as follows. 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 Streptomyces rapamycinicus, choose the double cross-over strain and test the fermentation
+ yield of rapamycin by HPLC.
+ </p>
+ <div class="imager">
+ <img class="rw-50"
+ src="https://static.igem.wiki/teams/4281/wiki/implementation/t-ecnuas-implementation-02.png" alt="">
+ <span class="figure">
+ <i>â–³M271_14685/m271_14690 and NRRL 5491 have produced rapamycin during 11 days.</i>
+ </span>
+ </div>
+ <p>
+ It can be seen from the above figure that the rapamycin produced by our knockout â–³M271_14685/m271_14690 is
+ much higher than that produced by NRRL 5491. And the amount of rapamycin produced on the ninth day reached
+ 120mg/L. This suggests that the subject is feasible to improve metabolic pathways by knocking out the
+ two-component system, which can be applied for clinical application in the near future.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-blue">3. Target Customer</h2>
+ <p>
+ The target users of our products are mainly patients who suffer from diseases requiring organ transplantation
+ and their families. As shown in the picture below, organ transplantation organizations are mainly located in
+ relatively developed provinces, such as Guangdong, Beijing, and Shanghai. Thus, on the basis of geographical
+ segmentation, it can be assumed that patients who mainly come from these regions could be our clients. And
+ since the most prominent competitive advantage of our product is the low price we set, from the perspective of
+ psychographic segmentation, patients who are sensitive to price are also our potential customers.
+ </p>
+ <div class="imager">
+ <img class="rw-75"
+ src="https://static.igem.wiki/teams/4281/wiki/implementation/t-ecnuas-implementation-03.png" alt="">
+ <span class="figure">
+ <i>Reginal distribution of organ transplantation medical organization in 2019 in China<sup>[5]</sup></i>
+ </span>
+ </div>
+ </section>
+
+ <section>
+ <h2 class="c-blue">4. Safety and Challenges</h2>
+ <p>
+ The whole process of our team's experiments and human practice activities fully comply with iGem safety
+ guidelines, both in terms of biosecurity and the Covid-19 situation.
+ </p>
+ <p>
+ In the vast majority of experiments, we used some safe and common laboratory strains, such as competent cells
+ <i>Escherichia coli</i> DH5α, competent cells <i>Escherichia coli</i> ET12567/pUZ8002, wild-type <i>Streptomyces
+ rapamycinicus</i>
+ NRRL 5491 and mutant strain ΔM271_14685/M271_14690. These are commonly used model organisms that have been
+ shown to be very safe, causing little or no harm to humans.
+ </p>
+ <p>
+ We have also fully considered the further safety issues that may arise in the future commercialization of the
+ product. Although with the help of biotechnology, the price we set can be lower than the competitors because
+ of the increase of production rate with lower costs. However, it should be noted that there are still several
+ potential challenges waiting to be addressed by us.
+ </p>
+ <ol class="ol-mark mark-quota-number-left l-start l-top-05 text-justify">
+ <li>The specific effects of our product have not been thoroughly examined for clinical trials have not been
+ conducted. To address the issue, our group plans to cooperate with local pharmaceutical enterprises,
+ healthcare companies, and hospitals to conduct further research and clinical trials to prevent any
+ unexpected side effects.
+ </li>
+ <li>The technology and product we adopt and produce have not passed the governmental audit to grant us with
+ the license to enter the pharmaceutical market. Hence, our group proposes to put the production process
+ under the supervision of the State Food and Drug Administration and strictly follow the relevant
+ governmental regulations.
+ </li>
+ <li>As a newly developed drug, our product can only be produced in the mode of low-volume production and brand
+ awareness will be low in the foreseeable future. Thus, marketing campaigns that aim to increase brand
+ exposure should be developed for our target customers.
+ </li>
+ </ol>
+ </section>
+
+ <section>
+ <h2 class="c-blue">5. Future Plan</h2>
+ <p>
+ As mentioned in the sections above, the main selling point of our product is the low price. Other than that,
+ as an injectable product, it is more favorable than oral products for it is more effective and convenient. At
+ present, no injectable Rapamune or other types of immunosuppressive drugs has been developed, suggesting a
+ huge gap in the market.
+ </p>
+ <p>
+ As a start-up enterprise, our marketing plan is mainly to improve brand awareness. We plan to carry out free
+ or inexpensive consultations for potential patients of organ transplantation or those who are interested in
+ this aspect. During the consultation, we can objectively promote our products. Outdoor advertising near organ
+ transplantation medical organizations, such as ads on the nearest bus stop or MTR station, can also be
+ implemented.
+ </p>
+ <p>
+ Regarding product development, we will consult more experts to optimize the conditions for higher production
+ of Rapamune through our engineered Streptomyces rapamycinicus and let it can be applied to industrial scale
+ production in the near future.
+ </p>
+ </section>
+
+ <section>
+ <h2 class="c-blue">Reference</h2>
+ <ol type="1" class="l-top-05 text-justify">
+ <li>《ä¸å›½å™¨å®˜ç§»æ¤å‘展报告（2020）》</li>
+ <li>《ä¸å›½å™¨å®˜æ献ä¹æš–还寒》, éƒè·¯ç‘¶, China Youth Daily, 2016.12.21 (09)</li>
+ <li>《ä¸åŽäººæ°‘共和国2016年国民ç»æµŽå’Œç¤¾ä¼šå‘展统计公报》, Xinhuanet, 2017.03.01</li>
+ <li>刘æž, å¼ çŽ², å™èˆª, et al. Application and development of enhancement factor of liver regeneration as
+ immunosuppressant:, 2006
+ </li>
+ <li>《ä¸å›½å™¨å®˜ç§»æ¤å‘展报告（2019）》</li>
+ </ol>
</section>
</div>
--
GitLab