Skip to content
Snippets Groups Projects
Commit 7b987fca authored by Remy Moll's avatar Remy Moll
Browse files

small style fixes

parent ccd99148
Branches remoll/feature/final-style-fixes
No related tags found
No related merge requests found
Hide whitespace changes
Inline Side-by-side
Showing
with 152 additions and 151 deletions
wiki/components/figure.html
+ 1
− 1
View file @ 7b987fca
<div class="row justify-content-center">
<div class="col col-12 col-md-9 col-lg-6">
<div class="col col-12 col-md-8 col-xxl-6">
<figure {% if figure.ref_id %}id="{{ figure.ref_id }}"{% endif %}>
<img src="{{ figure.src }}" class="img-fluid mx-auto d-block">
<figcaption class="figure-caption text-center"><strong>Figure {{ figure.number }}:</strong> {{ figure.caption | safe }}</figcaption>
......
wiki/components/references.html
+ 3
− 8
View file @ 7b987fca
<hr>
<h3 id="references">
References:
<button class="btn btn-primary" type="button" data-bs-toggle="collapse" data-bs-target="#collapseReference" aria-expanded="false" aria-controls="collapseReference">
Show references
</button>
</h3>
<div class="row mb-2 collapse" id="collapseReference">
<h3 id="references">References:</h3>
<small>
<ol class="list-group list-group-numbered">
{% for reference in references %}
<li class="list-group-item" id="ref{{ loop.index }}">
......@@ -17,4 +12,4 @@
</li>
{% endfor %}
</ol>
</div>
</small>
\ No newline at end of file
wiki/layouts/landingpage.html
+ 2
− 2
View file @ 7b987fca
......@@ -6,11 +6,11 @@
<div class="row justify-content-center" style="margin-top: 10vh; margin-bottom: 8vh;">
<div class="col-10 col-lg-8 col-xl-6 rounded shadow p-4" id="title-container">
<p class="text-center fs-3" id="over-title">Introducing...</p>
<p class="text-center fs-3" >Introducing...</p>
<h1 class="title text-center">
{% block title_block %}{% endblock %}
</h1>
<p class="text-center fs-3" id="over-title">{% block lead %}{% endblock %}</p>
<p class="text-center fs-3" >{% block lead %}{% endblock %}</p>
</div>
</div>
</style>
......
wiki/pages/attributions.html
+ 3
− 3
View file @ 7b987fca
......@@ -47,7 +47,7 @@
</div>
</div>
<p class="text-center fs-5" id="over-title">
<p class="text-center fs-5" >
<ul>
<li><b>Prof. Dr. Martin Jinek</b>: Professor Jinek kindly offered the wet lab team all the space resources necessary to carry out their experiments. He also discussed experimental design with the team. </li>
<li><b>Prof. Dr. Markus Seeger</b>: Professor Seeger not only hosted students in his lab, providing the necessary material for experiments but also shared his expertise by giving insights and recommendations during the experimental planning. </li>
......@@ -71,7 +71,7 @@
<i class="fas fa-users-line fa-3x mb-4"></i>
</div>
</div>
<p class="text-center fs-5" id="over-title">
<p class="text-center fs-5" >
<ul>
<li><b>Cyril Zipfel</b>: Professor Zipfel supported the team in administrative organisation related to our project. </li>
<li><b>Shila Schneider</b>: Shila's experience was very welcome during the later stages of our project, where we started thinking about bringing a drug to the market. TWe discussed research & development, preclinical steps and clinical trials and the burdens that are associated with each of these steps.</li>
......@@ -98,7 +98,7 @@
<i class="fas fa-display fa-3x mb-4"></i>
</div>
</div>
<p class="text-center fs-5" id="over-title">
<p class="text-center fs-5" >
<li><b>Prof. Dr. med. Dr. phil. Gerhard Rogler</b>: Prof. Dr. Rogler met with the dry lab team to discuss the model as well as inflammarory bowel disease. He helped our dry lab not only understand the limitations and potentials of our own model, but also the status quo of IBD medications and research. </li>
<li><b>Prof. Dr. rer. nat. Martin Hausmann</b>: Prof. Dr. Hausmann met with the dry lab team to discuss the model. Similarly to our meeting with Prof. Dr. Rogler, the exchange was extremely insightful and helped us pick the right parameters. </li>
<li><b>Hielke Walinga</b>: He took a lot of time to help set up the wiki to be able to work on it locally and submit the code from sublime text to the gitlab page. </li>
......
wiki/pages/description.html
+ 22
− 26
View file @ 7b987fca
......@@ -27,8 +27,11 @@
</div>
</div>
<p class="text-center">
Inflammatory Bowel Disease (IBD) comprises conditions in which the lining of the gastrointestinal tract is chronically inflamed leading to symptoms like diarrhea, abdominal pain, bloody stools and fatigue. The exact aetiology of IBD is a topic of extensive research whereby first hints point towards a multifactorial pathogenesis. [1](#references) Current treatment options are systemic and aim at modulating the immune system causing severe side effects. We, the UZurich iGEM Team, want to tackle the lack of targeted treatment options by developing a new microbe based therapy: IBD NanoBiotics. We modified Escherichia coli Nissle 1917 by introducing a genetic circuit that can produce and secrete anti tumor necrosis factor (TNF&alpha;) nanobodies upon selective induction by nitric oxide (NO) at the inflammation site. Along with our laboratory research, we have developed a spatial model and dedicated ourselves to outreach and education with a special focus on IBD patients.
<p class="text-center">
{% filter markdown %}
Inflammatory Bowel Disease (IBD) comprises conditions in which the lining of the gastrointestinal tract is chronically inflamed leading to symptoms like diarrhea, abdominal pain, bloody stools and fatigue. The exact aetiology of IBD is a topic of extensive research whereby first hints point towards a multifactorial pathogenesis. [1](#ref1) Current treatment options are systemic and aim at modulating the immune system causing severe side effects. We, the UZurich iGEM Team, want to tackle the lack of targeted treatment options by developing a new microbe based therapy: IBD NanoBiotics. We modified Escherichia coli Nissle 1917 by introducing a genetic circuit that can produce and secrete anti tumor necrosis factor (TNF&alpha;) nanobodies upon selective induction by nitric oxide (NO) at the inflammation site. Along with our laboratory research, we have developed a spatial model and dedicated ourselves to outreach and education with a special focus on IBD patients.
{% endfilter %}
<div class="row justify-content-center">
<div class="col-12 col-lg-9">
......@@ -45,30 +48,23 @@
---
### IBD: Crohn's disease and Ulcerative colitis
#### What is IBD?
<div class="row mt-4 align-items-center">
<div class="col col-12 col-lg-6">
{% filter markdown %}
Worldwide, over 6 million people are suffering from Inflammatory Bowel Disease (IBD) [5](#references) caused by chronic intestinal inflammation and tissue destruction triggered by an aberrant immune response. The term IBD comprises mainly two conditions: Crohn’s disease (CD) and Ulcerative colitis (UC). Patients diagnosed with CD might show inflammation throughout the entire gastrointestinal tract (GIT) while in patients that suffer from UC the inflammation is localized mainly in the colon and is continuous. As of today, the exact cause for IBD is still unknown. Research on the exact pathogenesis of IBD suggests a multifactorial mechanism where genetic, gut microbial and environmental factors are involved. [1](#references) The highest prevalence of IBD has been reported in Europe and Northern America with increasing incidence in newly industrialized countries. [2](#references) Patients suffering from IBD experience symptoms like diarrhea, bloody stools, and abdominal pain. However, symptoms are not confined to the inflamed areas and patients often suffer from other extraintestinal manifestations including mental health problems. Therapeutics such as aminosalicylates, corticosteroids, immunomodulators and inhibitory monoclonal antibodies are being used to alleviate the symptoms. However, these treatment options are known to cause systemic side effects and several cases of therapy resistance have been reported. [3](#references) Therefore, the need to develop a novel specific targeted treatment option causing less side effects is of utmost importance.
Worldwide, over 6 million people are suffering from Inflammatory Bowel Disease (IBD) [5](#ref5) caused by chronic intestinal inflammation and tissue destruction triggered by an aberrant immune response. The term IBD comprises mainly two conditions: Crohn’s disease (CD) and Ulcerative colitis (UC). Patients diagnosed with CD might show inflammation throughout the entire gastrointestinal tract (GIT) while in patients that suffer from UC the inflammation is localized mainly in the colon and is continuous. As of today, the exact cause for IBD is still unknown. Research on the exact pathogenesis of IBD suggests a multifactorial mechanism where genetic, gut microbial and environmental factors are involved. [1](#ref1) The highest prevalence of IBD has been reported in Europe and Northern America with increasing incidence in newly industrialized countries. [2](#ref2) Patients suffering from IBD experience symptoms like diarrhea, bloody stools, and abdominal pain. However, symptoms are not confined to the inflamed areas and patients often suffer from other extraintestinal manifestations including mental health problems. Therapeutics such as aminosalicylates, corticosteroids, immunomodulators and inhibitory monoclonal antibodies are being used to alleviate the symptoms. However, these treatment options are known to cause systemic side effects and several cases of therapy resistance have been reported. [3](#ref3) Therefore, the need to develop a novel specific targeted treatment option causing less side effects is of utmost importance.
{% endfilter %}
</div>
<div class="col col-12 col-lg-6">
{% with figure = {"src": "https://static.igem.wiki/teams/4387/wiki/whatsapp-image-2022-10-10-at-4-37-49-pm.jpeg", "number": "1", "caption": "Schematic of the different IBD pathology in CD and UC. Created with BioRender.com."} %}
{# replaced with bigger format instead of predefined figure#}
<figure {% if figure.ref_id %}id="{{ figure.ref_id }}"{% endif %}>
<img src="{{ figure.src }}" class="img-fluid mx-auto d-block w-75">
<figcaption class="figure-caption text-center"><strong>Figure 1:</strong> {{ figure.caption}}</figcaption>
</figure>
{% endwith %}
</div>
</div>
{% endfilter %}
{% with figure = {"src": "https://static.igem.wiki/teams/4387/wiki/whatsapp-image-2022-10-10-at-4-37-49-pm.jpeg", "number": "1", "caption": "Schematic of the different IBD pathology in CD and UC. Created with BioRender.com."} %}
{# replaced with bigger format instead of predefined figure#}
<figure {% if figure.ref_id %}id="{{ figure.ref_id }}"{% endif %}>
<img src="{{ figure.src }}" class="img-fluid mx-auto d-block w-75">
<figcaption class="figure-caption text-center"><strong>Figure 1:</strong> {{ figure.caption}}</figcaption>
</figure>
{% endwith %}
{% filter markdown %}
### Molecular pathology
In a healthy individual, the epithelial cells of the GIT are perfectly aligned to form an impermeable barrier which bacteria from the lumen cannot cross. Histologically, cells are found tightly bound to each other. [2](#references)
However, in patients suffering from IBD, this epithelial barrier and the protective mucus layer is disrupted, spaces between the cells arise and bacteria from inside the gut can invade the mucosa where they encounter immune cells, initiating inflammation. Inflammation then further damages the epithelial layer, allowing for more bacteria to penetrate the gut wall, increasing the inflammation levels even more. [2](#references) A schematic of the intestinal pathology is shown in figure 2. Very often, IBD is a life-long ongoing vicious cycle of alternating active inflammation and remission phases. [2](#references)
In a healthy individual, the epithelial cells of the GIT are perfectly aligned to form an impermeable barrier which bacteria from the lumen cannot cross. Histologically, cells are found tightly bound to each other. [2](#ref2)
However, in patients suffering from IBD, this epithelial barrier and the protective mucus layer is disrupted, spaces between the cells arise and bacteria from inside the gut can invade the mucosa where they encounter immune cells, initiating inflammation. Inflammation then further damages the epithelial layer, allowing for more bacteria to penetrate the gut wall, increasing the inflammation levels even more. [2](#ref2) A schematic of the intestinal pathology is shown in figure 2. Very often, IBD is a life-long ongoing vicious cycle of alternating active inflammation and remission phases. [2](#ref2)
{% endfilter %}
{% with figure = {"src": "https://static.igem.wiki/teams/4387/wiki/healthygut-vs-disease.jpg", "number": "2", "caption": "Scheme of a healthy gut wall vs. gut wall of an IBD patient. <i>Created with BioRender.com </i>"} %}
......@@ -91,18 +87,18 @@ We, the iGEM team 2022 from the University of Zurich, want to tackle the lack of
---
### Our engineering chassis
E. coli Nissle 1917 is a probiotic bacteria that can be found as a member of the human microbiota. It was first discovered in 1917 during the First World War from the German medical doctor Alfred Nissle in a soldier who did not show any symptoms after being exposed to the Shigella bacterium, known to cause diarrhea and fever. Dr. Nissle found out that the E. coli strain, which he previously isolated from the soldier’s stool, could prevent the spread of Shighella bacteria on petri dishes. [6](#references)
E. coli Nissle 1917 is a probiotic bacteria that can be found as a member of the human microbiota. It was first discovered in 1917 during the First World War from the German medical doctor Alfred Nissle in a soldier who did not show any symptoms after being exposed to the Shigella bacterium, known to cause diarrhea and fever. Dr. Nissle found out that the E. coli strain, which he previously isolated from the soldier’s stool, could prevent the spread of Shighella bacteria on petri dishes. [6](#ref6)
#### Tumor necrosis factor (TNF): an important mediator of inflammation
The TNF&alpha; molecule is a crucial regulator for the immune system's inflammatory response. It is mainly secreted by monocytes, macrophages, and T-cells and impacts the proliferation and differentiation of various cell typs. Additionally, it stimulates the production of further inflammatory cytokines and chemokines, enhancing the already existing inflammation. In the inflamed areas of the intestine, TNF&alpha; recruits immune cells causing intestinal fibrosis. [2](#references) Blocking the action of TNF&alpha; is already the goal of approved anti-TNF&alpha; therapies for IBD. However, it is known that patients receiving anti-TNF&alpha; biologics, such as the monoclonal antibodies infliximab or adalimumab, develop after a certain time of consistant exposure an antibody resistance which drastically reduces the efficacy of the therapy. Suppression of the immune system or switching to an alternative treatment option are unavoidable and further increase the discomfort of IBD patients. [8](#references)
The TNF&alpha; molecule is a crucial regulator for the immune system's inflammatory response. It is mainly secreted by monocytes, macrophages, and T-cells and impacts the proliferation and differentiation of various cell typs. Additionally, it stimulates the production of further inflammatory cytokines and chemokines, enhancing the already existing inflammation. In the inflamed areas of the intestine, TNF&alpha; recruits immune cells causing intestinal fibrosis. [2](#ref2) Blocking the action of TNF&alpha; is already the goal of approved anti-TNF&alpha; therapies for IBD. However, it is known that patients receiving anti-TNF&alpha; biologics, such as the monoclonal antibodies infliximab or adalimumab, develop after a certain time of consistant exposure an antibody resistance which drastically reduces the efficacy of the therapy. Suppression of the immune system or switching to an alternative treatment option are unavoidable and further increase the discomfort of IBD patients. [8](#ref8)
#### Nitric oxide as an inflammation marker
Our modified E. coli Nissle 1917 is able to sense the inflammation locally through a genetic circuit that responds to nitric oxide (NO) whose level is increased near inflammation sites. [2](#references) Moreover, this molecule is able to diffuse through the bacterial cell wall making the use of a special surface receptor irrelevant. Since the NO concentration is significantly elevated in inflammed tissue and NO shows the ability to diffuse through the bacterial cell wall, we focused on the design of a NO-sensor using the recently described pNorVβ promoter [4](#references), coupled to a positive feed-back loop with NorR, its corresponding transcription factor.
Our modified E. coli Nissle 1917 is able to sense the inflammation locally through a genetic circuit that responds to nitric oxide (NO) whose level is increased near inflammation sites. [2](#ref2) Moreover, this molecule is able to diffuse through the bacterial cell wall making the use of a special surface receptor irrelevant. Since the NO concentration is significantly elevated in inflammed tissue and NO shows the ability to diffuse through the bacterial cell wall, we focused on the design of a NO-sensor using the recently described pNorVβ promoter [4](#ref4), coupled to a positive feed-back loop with NorR, its corresponding transcription factor.
#### Nanobodies: potent single domain antibodies
Once sensed, NO will induce the production of nanobodies. Nanobodies, <!-- are recombinant 15 - 20 kDa small proteins originally isolated in camelids like alpacas or lamas. They consist of only one variable antibody domain and compared to antibodies are very stable and resistant to different temperature and pH conditions. Because of their small size, nanobodies can be produced and secreted to the extracellular environment from bacteria. For our project, we have worked with humanized anti-TNF&alpha; antibodies (PATENT) to avoid a possible immune reaction in vivo. We have coupled these nanobodies to a tag that allows their selective secretion via the hemolysin A secretion system, a type I secretion system found in uropathogenic E. coli strains. This one step secretion system consists of three proteins: TolC, hemolysin B (HlyB) and hemolysin D (HlyD). -->also called single domain antibodies or VHH, only consist of a single variable domain, which is able to bind to a specific antigen. The small size of nanobodies (15 - 20 kDa) gives them special abilities, which clearly distinct them from conventional antibodies. They can locally penetrate barriers (such as tissues) more easily and can withstand extreme environmental conditions, such as high temperatures and low pH. [9](#references) They show high affinity and stability, and recombinant expression has revolutionized the biotechnology field. Nanobodies have already been discovered in camelid animals back in the 90's. Usage of these nanobodies in the clinic often requires an additional step called "humanization" in order to reduce unwanted immunological reactions upon administration. This step describes the exchange of one or a few specific amino acids that are recognized as "foreign" by the human immune system. [10](#references) Still today, camelid animals are infected with the antigen of choice and effective nanobodies are obtained from their blood. However, new manufacturing technologies have been developed, allowing the screening of new candidates by using naive or synthetic libraries in combination with phage and ribosome display. The usage of synthetic libraries results in the generation of so called "sybodies". [11](#references)
Once sensed, NO will induce the production of nanobodies. Nanobodies, <!-- are recombinant 15 - 20 kDa small proteins originally isolated in camelids like alpacas or lamas. They consist of only one variable antibody domain and compared to antibodies are very stable and resistant to different temperature and pH conditions. Because of their small size, nanobodies can be produced and secreted to the extracellular environment from bacteria. For our project, we have worked with humanized anti-TNF&alpha; antibodies (PATENT) to avoid a possible immune reaction in vivo. We have coupled these nanobodies to a tag that allows their selective secretion via the hemolysin A secretion system, a type I secretion system found in uropathogenic E. coli strains. This one step secretion system consists of three proteins: TolC, hemolysin B (HlyB) and hemolysin D (HlyD). -->also called single domain antibodies or VHH, only consist of a single variable domain, which is able to bind to a specific antigen. The small size of nanobodies (15 - 20 kDa) gives them special abilities, which clearly distinct them from conventional antibodies. They can locally penetrate barriers (such as tissues) more easily and can withstand extreme environmental conditions, such as high temperatures and low pH. [9](#ref9) They show high affinity and stability, and recombinant expression has revolutionized the biotechnology field. Nanobodies have already been discovered in camelid animals back in the 90's. Usage of these nanobodies in the clinic often requires an additional step called "humanization" in order to reduce unwanted immunological reactions upon administration. This step describes the exchange of one or a few specific amino acids that are recognized as "foreign" by the human immune system. [10](#ref10) Still today, camelid animals are infected with the antigen of choice and effective nanobodies are obtained from their blood. However, new manufacturing technologies have been developed, allowing the screening of new candidates by using naive or synthetic libraries in combination with phage and ribosome display. The usage of synthetic libraries results in the generation of so called "sybodies". [11](#ref11)
Because of their small size, nanobodies can be produced and secreted to the extracellular environment from bacteria. [12](#references) For our project, we have worked with humanized anti-TNF&alpha; antibodies [10](#references) to avoid a possible immune reaction in vivo. We have coupled these nanobodies to a tag that allows their selective secretion via the hemolysin A secretion system, a type I secretion system found in uropathogenic E. coli strains. This one step secretion system consists of three proteins: TolC, hemolysin B (HlyB) and hemolysin D (HlyD). Together, these three proteins build a continuous channel through which originally the HlyA toxin is secreted in a one-step manner. Interestingly, the secretion signal is not found on the N-terminal site as it is for most secretion tags, but instead it is found at the C-terminal end and the signal sequence is not removed during secretion. Scientists have identified the secretion signal and were able to secrete various proteins of different sizes with the hemolysin A secretion machinery. [12](#references)
Because of their small size, nanobodies can be produced and secreted to the extracellular environment from bacteria. [12](#ref12) For our project, we have worked with humanized anti-TNF&alpha; antibodies [10](#ref10) to avoid a possible immune reaction in vivo. We have coupled these nanobodies to a tag that allows their selective secretion via the hemolysin A secretion system, a type I secretion system found in uropathogenic E. coli strains. This one step secretion system consists of three proteins: TolC, hemolysin B (HlyB) and hemolysin D (HlyD). Together, these three proteins build a continuous channel through which originally the HlyA toxin is secreted in a one-step manner. Interestingly, the secretion signal is not found on the N-terminal site as it is for most secretion tags, but instead it is found at the C-terminal end and the signal sequence is not removed during secretion. Scientists have identified the secretion signal and were able to secrete various proteins of different sizes with the hemolysin A secretion machinery. [12](#ref12)
### IBD Nanobiotics: an engineered E.coli Nissle 1917 to fight inflammation in IBD patients
Summarized, the engineering of our chassis E. coli Nissle 1917 consists of:
......
wiki/pages/engineering.html
+ 11
− 13
View file @ 7b987fca
......@@ -59,7 +59,7 @@ Purified nanobodies were then tested for their ability to bind biotinylated TNF&
<div class="col-lg-6">
<figure>
<div style="width: fit-content; margin: auto">
<img src="https://static.igem.wiki/teams/4387/wiki/elisa-pur-nbs.jpg" class="img-fluid">
<img src="https://static.igem.wiki/teams/4387/wiki/elisa-pur-nbs.jpg" class="img-fluid p-1">
</div>
<figcaption class="figure-caption text-center"> <strong>Figure 1A</strong>: ELISA performed with all purified nanobody constructs. One bivalent nanobody (N7 = linked VHH#3E + VHH#2B) is missing. First four wells show the negative and postive control.</figcaption>
</figure>
......@@ -206,20 +206,18 @@ We are able to show with these results that an induced secretion of functional a
{% endfilter %}
<hr>
<div class="row mt-4">
<div class="col", id = "references">
<h2>References</h2>
{% filter markdown %}
* [1] Qingdong Guan, 2019, “A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Diseas”
* [2] Silence, Karen, Lauwereys, Marc, De Haard, Hans, et al. "Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor", Int. Publication Number: WO 2004/041862 A2, 21 May 2004
* [3] Xiaoyu J. Chen et al., 2021, Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells
* [4] Ruano-Gallego, D., Fraile, S., Gutierrez, C. et al. Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact 18, 47 (2019)
{% endfilter %}
</div>
</div>
{% set references = [
{"name": 'Qingdong Guan, 2019, “A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Diseas”'},
{"name": 'Silence, Karen, Lauwereys, Marc, De Haard, Hans, et al. "Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor", Int. Publication Number: WO 2004/041862 A2, 21 May 2004'},
{"name": 'Xiaoyu J. Chen et al., 2021, Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells'},
{"name": 'Ruano-Gallego, D., Fraile, S., Gutierrez, C. et al. Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact 18, 47 (2019)'},
] %}
{% with references = references %}
{% include "components/references.html" %}
{% endwith %}
......
wiki/pages/entrepreneurship.html
+ 23
− 12
View file @ 7b987fca
......@@ -6,11 +6,11 @@
{% block lead %}{% endblock %}
{% block page_content %}
<div class="row justify-content-center px-3 mt-5">
<div class="card shadow-sm" style="width: 18rem;">
<figure>
<blockquote class="blockquote">
<p class="text-center">" Know the value of your asset as it will depend on where in this market you want to position."</p>
<div class="row justify-content-center mt-3">
<div class="card shadow px-3 pt-3" style="width: 18rem;">
<figure>
<blockquote class="blockquote">
<p class="text-center">" Know the value of your asset as it will depend on where in this market you want to position."</p>
</blockquote>
<figcaption class="blockquote-footer text-center">
Stakeholder</cite>
......@@ -38,10 +38,11 @@ By utilizing synthetic biology's tools, we came up with the [pioneering idea](ht
* Decrease systemic side effects and reduce annual physician visits related to them
In figure 1, you see the SWOT analysis for IBD NanoBiotics. It’s integral to know, where we stand and what our strengths, but also weaknesses are.
{% endfilter %}
{% with figure = {"src": "https://static.igem.wiki/teams/4387/wiki/swot-analysis.png", "number": "1", "caption": "SWOT analysis showing the strengths and risks."} %}
{% include "components/figure.html"%}
{% endwith %}
{% filter markdown %}
## Global Market Analysis
The global market for Inflammatory Bowel Disease treatment was valued at 23.9 billion US Dollars in 2021. [5](#references) Projections predict a Compound Annual Growth Rate (CAGR) of 5.7%, leading to a market size of 37 billion US Dollars by 2029. [5](#references) Therefore, we expect the demand for our product to increase as well, making it a promising investment option for future stakeholders.
......@@ -57,11 +58,11 @@ Applying for a patent would mean that all the project components should be ready
Our collaboration with Pharmabiome, an ETH spin-off, has been fundamental to understanding and dissecting the market of microbiome-based therapies. As they are also at an early market stage, they provided us with fundamental insight and expertise. We are thrilled to see what the future holds for IBD NanoBiotics!
<div class="row justify-content-center px-3 mt-5">
<div class="card shadow-sm" style="width: 18rem;">
<figure>
<blockquote class="blockquote">
<p class="text-center">"...taking more risks means getting the biggest share out of it.."</p>
<div class="row justify-content-center my-3">
<div class="card shadow px-3 pt-3" style="width: 18rem;">
<figure>
<blockquote class="blockquote">
<p class="text-center">"...taking more risks means getting the biggest share out of it.."</p>
</blockquote>
<figcaption class="blockquote-footer text-center">
Business Development manager</cite>
......@@ -70,10 +71,20 @@ Our collaboration with Pharmabiome, an ETH spin-off, has been fundamental to und
</div>
</div>
## References
{%endfilter%}
{% set references = [
{"url": "https://doi.org/10.1186/s12876-017-0681-y", "name": "Bähler, C., Vavricka, S. R., Schoepfer, A. M., Brüngger, B., & Reich, O. (2017). Trends in prevalence, mortality, health care utilization and health care costs of Swiss IBD patients: a claims data based study of the years 2010, 2012 and 2014. BMC gastroenterology, 17(1), 138. "},
{"url": "https://doi.org/10.1155/2019/7247238", "name": "Guan Q. (2019). A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Disease. Journal of immunology research, 2019, 7247238."},
{"url": "https://doi.org/10.3389/fmed.2021.765474", "name": "Cai, Z., Wang, S., & Li, J. (2021). Treatment of Inflammatory Bowel Disease: A Comprehensive Review. Frontiers in medicine, 8, 765474."},
{"url": "https://doi.org/10.1097/MEG.0000000000001616", "name": "Schoepfer, A., Vavricka, S. R., Brüngger, B., Blozik, E., & Bähler, C. (2020). Systematic analysis of therapeutic patterns and healthcare use during 12 months before inflammatory bowel disease-related hospitalization in Switzerland. European journal of gastroenterology & hepatology, 32(3), 350–357."},
{"url": "https://www.fortunebusinessinsights.com/inflammatory-bowel-disease-treatment-market-106704", "name": 'Fortune Business Insights, May 2022. "The global inflammatory bowel disease treatment market is projected to grow from $25.18 billion in 2022 to $37.00 billion by 2029, at a CAGR of 5.7%..."'},
] %}
{% with references = references %}
{% include "components/references.html" %}
{% endwith %}
<div style="max-width: 100;", id = "references">
<ol type="1">
<add-reference
......
wiki/pages/home.html
+ 9
− 9
View file @ 7b987fca
......@@ -92,23 +92,23 @@ Inflammatory Bowel Disease (IBD) is an umbrella term that comprises conditions s
<div class="row row-cols-1 row-cols-md-3 justify-content-center align-items-center px-5 py-5 g-5">
<div class="col justify-content-center text-center">
<i class="fas fa-house-medical fa-3x mb-4"></i>
{% filter markdown %}
**1** in **250** people in Switzerland suffer from IBD
{% endfilter %}
<p class="text-center">
<strong>1</strong> in <strong>250</strong> people in Switzerland suffer from IBD
</p>
</div>
<div class="col justify-content-center text-center">
<i class="fas fa-coins fa-3x mb-4"></i>
{% filter markdown %}
**7.8 x** higher costs on Swiss health care system than non IBD-patients
{% endfilter %}
<p class="text-center">
<strong>7.8 x</strong> higher costs on Swiss health care system than non IBD-patients
</p>
</div>
<div class="col justify-content-center text-center">
<i class="fas fa-pills fa-3x mb-4"></i>
{% filter markdown %}
**4** drug classes used only to manage symptoms
{% endfilter %}
<p class="text-center">
<strong>4</strong> drug classes used only to manage symptoms
</p>
</div>
</div>
</section>
......
wiki/pages/results.html
+ 78
− 77
View file @ 7b987fca
......@@ -28,35 +28,38 @@
<div class="row justify-content-center" style="margin-top: 10vh; margin-bottom: 8vh;">
<div class="titledivcolor col-10 col-lg-8 col-xl-12 rounded shadow p-4" id="title-container">
<div style="width: 100%; margin-left: auto; margin-right: auto">
<div style="width: 100%; display: inline-flex; justify-content: space-between;">
<h1 class="title text-center"> Achievements</h1>
<i class="fas fa-microscope fa-3x mb-4"></i>
</div>
</div>
<p class="text-center fs-5" id="over-title">
<ul>
<b>Wet lab</b>
<li>Production and purification of 3 monovalent and 6 bivalent nanobody constructs</li>
<li>Confirmation of the inhibitory effect of purified anti-TNF&alpha; nanobodies on inflammatory cell signalling</li>
<li>Arabinose-induced secretion of nanobodies in E. coli Nissle 1917 and proof of efficacy</li>
<li>Confirmation of the sensitivity of pNorV&beta; to nitric oxide</li>
<!--<li>Confirmation that pNorV&beta; induces higher responses to nitric oxide than the precedently characterized part from the ETH team 2016</li>-->
<li>Building of an optimised nitric oxide sensing system capable of inducing high GFP expression after induction and with better sensitivity to nitric oxide than the ETH promoter (EC50 = 335 &mu;M)</li>
<li>Final Product: NO-induced secretion of nanobodies in E. coli Nissle 1917 and proof of efficacy</li>
<b> Dry lab</b>
<li>Simulation of Nanobody treatment efficacy</li>
<li>Emulation of diseased gut environment with all relevant particles and their interactions</li>
<li>Approximation to continous time with adapted diffusion, emission and decay patterns</li>
<li>High-throughput parameter search for range of successful treatments</li>
<li>Backbone model made available for further use in IBD research and all particle emission & diffusion systems</li>
</ul>
</p>
<div style="width: 100%; display: inline-flex; justify-content: space-between;">
<h1 class="title text-center"></h1>
<i class="fas fa-display fa-3x mb-4"></i>
</div>
<div style="width: 100%; margin-left: auto; margin-right: auto">
<div style="width: 100%; display: inline-flex; justify-content: space-between;">
<h1 class="title text-center"> Achievements</h1>
<i class="fas fa-microscope fa-3x mb-4"></i>
</div>
</div>
<p class="text-center fs-5"><b>Wet lab</b></p>
<ul>
<li>Production and purification of 3 monovalent and 6 bivalent nanobody constructs</li>
<li>Confirmation of the inhibitory effect of purified anti-TNF&alpha; nanobodies on inflammatory cell signalling</li>
<li>Arabinose-induced secretion of nanobodies in E. coli Nissle 1917 and proof of efficacy</li>
<li>Confirmation of the sensitivity of pNorV&beta; to nitric oxide</li>
<!--<li>Confirmation that pNorV&beta; induces higher responses to nitric oxide than the precedently characterized part from the ETH team 2016</li>-->
<li>Building of an optimised nitric oxide sensing system capable of inducing high GFP expression after induction and with better sensitivity to nitric oxide than the ETH promoter (EC50 = 335 &mu;M)</li>
<li>Final Product: NO-induced secretion of nanobodies in E. coli Nissle 1917 and proof of efficacy</li>
</ul>
<p class="text-center fs-5 mt-2"><b>Dry lab</b></p>
<ul>
<li>Simulation of Nanobody treatment efficacy</li>
<li>Emulation of diseased gut environment with all relevant particles and their interactions</li>
<li>Approximation to continous time with adapted diffusion, emission and decay patterns</li>
<li>High-throughput parameter search for range of successful treatments</li>
<li>Backbone model made available for further use in IBD research and all particle emission & diffusion systems</li>
</ul>
<div style="width: 100%; display: inline-flex; justify-content: space-between;">
<h1 class="title text-center"></h1>
<i class="fas fa-display fa-3x mb-4"></i>
</div>
</div>
</div>
......@@ -68,8 +71,8 @@ The wet lab team was able to engineer our intended end chassis, the probiotic E.
All of our plasmid maps are available [here](https://2022.igem.wiki/uzurich/engineering#plasmids).
A detailed methods and protocols section can be found [here](https://2022.igem.wiki/uzurich/experiments).
<hr>
---
### Induction of pNorV&beta;:
**Aim**: Our aim was to create a biosensor for the inflammation marker nitric oxide (NO).
......@@ -128,14 +131,11 @@ From Figure 2a, it also seems that the construct with 2 ribosome binding sites i
In order to do this, we removed NorR from our construct by Gibson assembly and re-ran the same plate-reader experiment. However, this did not improve our range and reduced the GFP expression.
<div class="row mt-4 justify-content-center">
<figure>
<div style="width: fit-content; margin: auto">
<img src="https://static.igem.wiki/teams/4387/wiki/no-graphs/comparison-nono-new.jpeg" class="img-fluid mx-auto", width = "80%">
</div>
<figcaption class="figure-caption text-center"><strong>Figure 3</strong>: Response of our construct with 2 RBSs to induction with DETA/NO.</figcaption>
</figure>
</div>
{% endfilter %}
{% with figure = {"src": "https://static.igem.wiki/teams/4387/wiki/no-graphs/comparison-nono-new.jpeg", "number": "3", "caption": "Response of our construct with 2 RBSs to induction with DETA/NO."} %}
{% include "components/figure.html"%}
{% endwith %}
{% filter markdown %}
Due to time constrains, we were not able to further try to adjust the sensing range and decided to continue with our previous construct (our genetic circuit with 2 RBSs), which seemed to give us the highest GFP expression.
......@@ -247,12 +247,13 @@ Trying the same procedure with pOSIP-KO ordered from Addgene, we were able to tr
</figure>
Afterwards, we tested the binding ability to TNF&alpha; of the purified nanobodies with an ELISA.
<figure>
<img src="https://static.igem.wiki/teams/4387/wiki/elisa-pur-nbs.jpg" class="img-fluid">
<figcaption class="figure-caption text-center"><strong>Figure 9</strong>: ELISA performed with all purified nanobody constructs. One bivalent nanobody (N7 = linked VHH#3E + VHH#2B) is missing. First four wells show the negative and positive control.</figcaption>
</figure>
{% endfilter %}
{% with figure = {"src": "https://static.igem.wiki/teams/4387/wiki/elisa-pur-nbs.jpg", "number": "9", "caption": "ELISA performed with all purified nanobody constructs. One bivalent nanobody (N7 = linked VHH#3E + VHH#2B) is missing. First four wells show the negative and positive control."} %}
{% include "components/figure.html"%}
{% endwith %}
{% filter markdown %}
**Conclusion**: Monovalent and bivalent nanobody constructs targeting TNF&alpha; can be expressed and extracted from E. coli MC1061. Additionally these nanobodies effectively bind TNF&alpha; as shown with an ELISA.
### Inhibition of TNF&alpha; signalling using purified anti-TNF&alpha; nanobodies (cell assays)
......@@ -261,13 +262,13 @@ Afterwards, we tested the binding ability to TNF&alpha; of the purified nanobodi
We used the human monocytic cell line THP-1 to show that inhibiting TNF&alpha;-actions influences the immune response of human monocytes. For this, we incubated first the monocytes with different nanobody constructs and then stimulated the cells for 24 hours with different concentrations of human recombinant TNF&alpha;. We then measured the inflammatory response of the cells indirectly by comparing the IL-1&beta; expression to the housekeeping gene GAPDH. IL-1&beta; is an important inflammation mediator and therefore a good marker to prove functional TNF&alpha;-inhibition. Adalimumab is a commercially available anti-TNF&alpha; monoclonal antibody already used clinically to treat IBD patients. It served as a positive control in our assays.
<figure>
<div style="width: fit-content; margin: auto">
<img src="https://static.igem.wiki/teams/4387/wiki/all-nbs-cell-assay.png" class="img-fluid mx-auto" width = "80%">
</div>
<figcaption class="figure-caption text-center"><strong>Figure 10</strong>: IL-1&beta; expression compared to human GAPDH in THP-1 cells. Cells were stimulated with different TNF&alpha; concentrations. The nanobodies were purified from E. coli MC1061. Nb1 = VHH#2B, Nb2 = VHH#3E, Nb3 = VHH#12B, Nb9 = VHH#3E + VHH#12B</figcaption>
</figure>
{% endfilter %}
{% with figure = {"src": "https://static.igem.wiki/teams/4387/wiki/all-nbs-cell-assay.png", "number": "10", "caption": "IL-1&beta; expression compared to human GAPDH in THP-1 cells. Cells were stimulated with different TNF&alpha; concentrations. The nanobodies were purified from E. coli MC1061. Nb1 = VHH#2B, Nb2 = VHH#3E, Nb3 = VHH#12B, Nb9 = VHH#3E + VHH#12B"} %}
{% include "components/figure.html"%}
{% endwith %}
{% filter markdown %}
**Conclusion**: Our analysis showed that TNF&alpha; alone induces a significant expression of IL-1&beta; and all nanobodies, monovalent and bivalent constructs, were able to reduce the inflammatory response by up to 4-fold. Additionally, most nanobodies performed as good or even better than the monoclonal antibody adalimumab.
### Production and secretion of nanobodies in E. coli MC1061
......@@ -335,17 +336,19 @@ We simulated the gut environment and all relevant particles. The backbone of the
###Results
When applying the model to the scope of IBD, we can see in figure 13 that no matter the severity of inflammation, the nanobodies will lead to an exponential decay of TNFα and eventually converge.
<div class="imageBox">
<figure>
<div>
<img src="https://static.igem.wiki/teams/4387/wiki/image-6.png" class="img-fluid">
<figcaption class="figure-caption text-center"><strong>Figure 14: </strong>TNF&alpha; reduction for different amounts of inflammation sites </figcaption>
</div>
<div>
<img src="https://static.igem.wiki/teams/4387/wiki/image-8.png" class="img-fluid">
<figcaption class="figure-caption text-center"> <strong>Figure 15: </strong>TNF&alpha; reduction for different nanobody production efficacies</figcaption>
</div>
</figure>
<div class="row row-cols-1 row-cols-md-2 g-2">
<div class="col">
<figure>
<img src="https://static.igem.wiki/teams/4387/wiki/image-6.png" class="img-fluid">
<figcaption class="figure-caption text-center"><strong>Figure 14: </strong>TNF&alpha; reduction for different amounts of inflammation sites </figcaption>
</figure>
</div>
<div class="col">
<figure>
<img src="https://static.igem.wiki/teams/4387/wiki/image-8.png" class="img-fluid">
<figcaption class="figure-caption text-center"> <strong>Figure 15: </strong>TNF&alpha; reduction for different nanobody production efficacies</figcaption>
</figure>
</div>
</div>
More surprising is that the results of figure 15 seem to be invariant with the production of Nanobodies from a single bacteria, enabling future research to focus on the viability of the bacteria. Furthermore, we can tip the trade-off between competitiveness and Nanobody production further toward the survival of the bacteria.
......@@ -375,18 +378,16 @@ More surprising is that the results of figure 15 seem to be invariant with the p
{% endfilter %}
<div class="row mt-4">
<div class="col", id = "references">
<h2>References</h2>
{% filter markdown %}
* [1] Xiaoyu J. Chen et al., 2021, Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells
* [2] Cui L. et al, 2017, Clonetegration using OSIP plasmids: One-step DNA assembly and site-specific genomic integration in bacteria
* [3] Rottinghaus, Austin G. et al, 2022, Genetically stable CRISPR-based kill switches for engineered
* [4] Ulrich Sonnenborn, 2017, "100 years of E. coli strain Nissle 1917", Oxford University Press's blog, https://blog.oup.com/2017/04/e-coli-strain-nissle-1917-timeline/
{% endfilter %}
</div>
</div>
{% set references = [
{"name": 'Xiaoyu J. Chen et al., 2021, Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells'},
{"name": 'Cui L. et al, 2017, Clonetegration using OSIP plasmids: One-step DNA assembly and site-specific genomic integration in bacteria'},
{"name": 'Rottinghaus, Austin G. et al, 2022, Genetically stable CRISPR-based kill switches for engineered '},
{"url": "https://blog.oup.com/2017/04/e-coli-strain-nissle-1917-timeline/", "name": 'Ulrich Sonnenborn, 2017, "100 years of E. coli strain Nissle 1917", Oxford University Press blog'},
] %}
{% with references = references %}
{% include "components/references.html" %}
{% endwith %}
{% endblock %}
0% or .
You are about to add 0 people to the discussion. Proceed with caution.
Finish editing this message first!
Please register or to comment