diff --git a/wiki/pages/collaborations.html b/wiki/pages/collaborations.html
index 14ce54930e1a2cd4adcd037793b1b40a6adf7ad2..f62531de0263afe59a226b8ea01f1b350028bae2 100644
--- a/wiki/pages/collaborations.html
+++ b/wiki/pages/collaborations.html
@@ -60,6 +60,10 @@
<p>The <a href="https://2022.igem.wiki/ubc-okanagan/" target="_blank">UBCO</a> iGEM team is a <span id="highlight">first-time team that we advised for the duration of the iGEM season</span> on team development, project ideation, and logistics. Our <span id="highlight">mentorship relationship</span> fostered an environment where we were comfortable sharing tips and asking questions related to team development, sponsorship, Wet-Lab work, and other collaborations. They provided the contact for Dr. Luis DeStefano, a speaker in our international accessibility panel, while we provided a contact for obtaining the bacterial strain for their project.</p>
<br>
<p>We also participated in their <span id="highlight">Phototroph Meetup conference</span>, where they fostered a community around working with phototrophs in iGEM given their challenges. The Phototroph community was started by the 2021 Marburg iGEM team to compile useful information for future iGEM teams working with phototrophic organisms to help fill the gap in available resources and protocols. After its success last year, the idea was continued by the UBCO team, and our team got the chance to join the community of 7 teams from around the world. As a part of the community, we participated in two <span id="highlight">virtual meetups</span> throughout July and August, where we gained insightful advice from academic experts and industry professionals working in plant synthetic biology. Each team within the community was given a chance to share their project and receive feedback from each other as well as experts. We utilized this opportunity to gain insightful troubleshooting advice for our protoplast isolation. As Synaestivum was the first plant-based project any of our members had worked on, being a part of the community and learning from the other teams was an integral part of our project development. We also added protocol documentation to their <span id="highlight">Phototroph Community Handbook</span>, meant to share methods and protocols related to working with phototrophs to increase documentation for future teams to work with them as well. We outlined the premise and troubleshooting related to our protoplast isolation procedure to increase the amount of teams that could work on this in the future.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/phototroph-meetup.png" alt="Phototroph Meetup Conference" width="80%">
+ <figcaption>Phototroph Community Meetup Conference</figcaption>
+ </figure>
<br>
<h2 id="Patras">Patras iGEM</h2>
<hr>
diff --git a/wiki/pages/communication.html b/wiki/pages/communication.html
index 3732718b1010fff7171e2b5dbdbbbef2e77f40de..c13c305a3f7639649fc759b37da60d1088fb97f6 100644
--- a/wiki/pages/communication.html
+++ b/wiki/pages/communication.html
@@ -50,7 +50,12 @@
<p>Through <span id="highlight">volunteering with the Yard Garden Harvest Project</span> with actions such as garden maintenance and interaction with the local community to raise awareness about the garden, we were able to increase food security in our vicinity, which we also hope to achieve with the completion of our project.</p>
<br>
<h5><b><u>Ensuring Responsible Production & Consumption</u></b></h5>
- <p>As outlined in our <a href="https://2022.igem.wiki/ubc-vancouver/sustainable" target="_blank">Sustainable Development Goals</a> page, we are happy that the development of our project contributes to <span id="highlight">sustainable production and consumption systems</span>. The premise of our project involves <span id="highlight">generating wheat for human consumption in a manner that produces less biomass loss</span>, therefore a more sustainably produced variety of wheat than one that would make poorer use of resources through increased biomass loss. Even though our project itself contributes to this SDG, a significant amount of effort towards reaching the SDGs is increasing education around them to encourage others to pursue them as well. As such, we wanted to raise awareness of admirable strategies individuals have implemented to address this SDG, and inspire others to do the same. For this reason, we <span id="highlight">launched our ongoing [PODCAST NAME], launching three episodes with representatives from both industry and academia</span> working on varying approaches to reach this SDG. We acknowledge that promoting this podcast within Canada might mostly reach already-educated individuals that are aware of the SDGs, while our goal is to increase awareness of what activities can be done to reach the SDGs to individuals who are less aware of them. As such, we have been actively encouraging the <span id="highlight">podcast's promotion across four continents through sharing it with the network created from our International Accessibility Panel</span> (details below).</p>
+ <p>As outlined in our <a href="https://2022.igem.wiki/ubc-vancouver/sustainable" target="_blank">Sustainable Development Goals</a> page, we are happy that the development of our project contributes to <span id="highlight">sustainable production and consumption systems</span>. The premise of our project involves <span id="highlight">generating wheat for human consumption in a manner that produces less biomass loss</span>, therefore a more sustainably produced variety of wheat than one that would make poorer use of resources through increased biomass loss. Even though our project itself contributes to this SDG, a significant amount of effort towards reaching the SDGs is increasing education around them to encourage others to pursue them as well. As such, we wanted to raise awareness of admirable strategies individuals have implemented to address this SDG, and inspire others to do the same. For this reason, we <span id="highlight">launched our ongoing podcast: "Sustainable Development in Synthetic Biology", launching three episodes with representatives from both industry and academia</span> working on varying approaches to reach this SDG. We acknowledge that promoting this podcast within Canada might mostly reach already-educated individuals that are aware of the SDGs, while our goal is to increase awareness of what activities can be done to reach the SDGs to individuals who are less aware of them. As such, we have been actively encouraging the <span id="highlight">podcast's promotion across four continents through sharing it with the network created from our International Accessibility Panel</span> (details below).</p>
+ <br>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/podcast.png" width="80%">
+ <figcaption>Podcast Links: RSS (<a href="https://rss.com/podcasts/ubcigem/" target="_blank">https://rss.com/podcasts/ubcigem/</a>) and <a href="https://open.spotify.com/show/0XoxgxlAEtypmucLuxUzig?si=f26944e25a004c89&nd=1" target="_blank">Spotify</a></figcaption>
+ </figure>
<br>
<h2 id="EndUserGoals">End-User Goals</h2>
<hr>
@@ -104,11 +109,11 @@
<div class="accordion" id="accordionExample">
<div class="accordion-item">
<h2 class="accordion-header" id="references">
- <button class="accordion-button" type="button" data-bs-toggle="collapse" data-bs-target="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
+ <button class="accordion-button collapsed" type="button" data-bs-toggle="collapse" data-bs-target="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
References
</button>
</h2>
- <div id="collapseOne" class="accordion-collapse collapse show" aria-labelledby="references" data-bs-parent="#accordionExample">
+ <div id="collapseOne" class="accordion-collapse collapse" aria-labelledby="references" data-bs-parent="#accordionExample">
<div class="accordion-body">
<p><span id='highlight'>[1]</span> Chugh, A. (2021). How can technological advancements in synthetic biology benefit everyone? An expert explains. <i>World Economic Forum.</i> <a href="https://www.weforum.org/agenda/2021/11/synthetic-biology-can-benefit-all-expert-explains-how/" target="_blank">https://www.weforum.org/agenda/2021/11/synthetic-biology-can-benefit-all-expert-explains-how/</a></p>
<p><span id='highlight'>[2]</span> Shisanya, S.O. & Hendriks, S.L. (2011). The contribution of community gardens to food security in the Maphephetheni uplands, determined by the Household Food Insecurity Access Scale. <i>Development Southern Africa, 28(4), 509-526.</i></p>
diff --git a/wiki/pages/education.html b/wiki/pages/education.html
new file mode 100644
index 0000000000000000000000000000000000000000..31b979192fc9e30b2e1304f8914228bba8c86971
--- /dev/null
+++ b/wiki/pages/education.html
@@ -0,0 +1,128 @@
+{% extends "layout.html" %}
+
+{% block lead %}HUMAN PRACTICES{% endblock %}
+{% block title %}Education{% endblock %}
+
+{% block page_content %}
+<html>
+ <head>
+ <style>
+ /* overwrite default banner photo for the Experiments pages*/
+ .bg-hero {
+ background-image: url('https://static.igem.wiki/teams/4296/wiki/banners/field5-wheatgrown-talking.jpg') !important;
+ background-size: cover;
+ }
+
+ figure img {
+ display: block;
+ margin: auto;
+ }
+ </style>
+ </head>
+ <body>
+ <div class="row">
+ <div class="sticky col-2">
+ <div class="sections">
+ <a class="section" href="#Outreach">Outreach, Education, & Communication</a>
+ <hr class="sidebar-hr">
+ <a class="section" href="#ProductDevelopment">Product Development Goals</a>
+ <hr class="sidebar-hr">
+ <a class="section" href="#EndUserGoals">End-User Goals</a>
+ </div>
+ </div>
+ <div class="content col-10">
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/hp-outreach.png" alt="Human Practices Outreach" width="100%">
+ <figcaption>UBC iGEM’s Outreach, Education, & Communication Objectives</figcaption>
+ </figure>
+ <br><br>
+ <h2 id="Outreach">Outreach, Education, & Communication</h2>
+ <hr>
+ <p>Though the technical work of our project makes up for its success, it is essential to make sure our work is <span id="highlight">responsible within our research and innovation</span>. This involves being aware of the different ways our project could impact individuals from different backgrounds, encouraging us to ensure that the intended purpose and use of our project is received correctly. As developers of a product, our team has a vision of the outcomes we want our end-users to obtain, but to be able to receive those benefits - in our case, <span id="highlight">increased food security and a way for citizens to contribute to sustainable production and consumption systems</span> - we must carry out additional efforts that enforce these values. Therefore, we have focused around half of our outreach projects on strengthening the outcomes we would like to evoke through <span id="highlight">education</span>.</p>
+ <br>
+ <p>While we ensure our project is technically sound, we must also emphasize its purpose and intent, in order for our target end-users to be ready to receive this novel innovation. The second half of our outreach activities are centered around <span id="highlight">making sure our audience can access our innovation</span> correctly. Since our product is centered around genetically modified foods, we want to <span id="highlight">ensure our end-users are as open as possible to the possibility of consuming GMOs</span>. Secondly, developing this project as a Canadian team puts us at a disadvantage of benefitting North American farmers, even though we are targeting small-scale farmers that are unable to recover from large yield losses of wheat throughout the world. While acceptability of synthetic biology innovations across the world is a topic we aim to address, there is also an inequality in how accessible it is to carry out synthetic biology processes due to lack of resources and infrastructure in certain countries<span id="highlight"><sup>[1]</sup></span>. This prevents countries from, for example, innovating and engineering agriculture in a similar manner that is more tailored to their needs if they are inspired from our project. Hence, we also wanted to <span id="highlight">increase accessibility to synthetic biology to carry across the world</span>.</p>
+ <br>
+ <h2 id="ProductDevelopment">Product Development Goals</h2>
+ <hr>
+ <h5><b><u>Food Security</u></b></h5>
+ <p>The main vision of our project is to <span id="highlight">increase food security by creating more reliable crops that have less yield variability in response to varying temperatures</span>. As such, we wanted to reflect this vision not only through the technical work our team was conducting, but also through aiding other efforts with this same goal, especially considering that enhancing wheat food security is a small piece in the context of the larger problem. In searching for <span id="highlight">local initiatives that aid food security of our community in crops</span> other than wheat, we came across the <a href="https://spec.bc.ca/yard-garden-harvest-project/" target="_blank">Yard Garden Harvest Project</a>, a community garden meant to increase food security. One of the main facets of food insecurity is the lack of access to nutritious foods due to their increased prices in comparison to less nutritious food. In addition to this, urbanization results in even more decreased access to fresh produce since it decreases opportunities to create personal gardening and agricultural practices for families. <span id="highlight">Community gardens</span> can battle this facet of food insecurity through offering free nutritious alternatives, making a balanced diet more accessible to the community<span id="highlight"><sup>[2]</sup></span>. This reaches the same goal as our project of being able to increase the “reliability†of certain food types in people's diets.</p>
+ <br>
+ <p>Through <span id="highlight">volunteering with the Yard Garden Harvest Project</span> with actions such as garden maintenance and interaction with the local community to raise awareness about the garden, we were able to increase food security in our vicinity, which we also hope to achieve with the completion of our project.</p>
+ <br>
+ <h5><b><u>Ensuring Responsible Production & Consumption</u></b></h5>
+ <p>As outlined in our <a href="https://2022.igem.wiki/ubc-vancouver/sustainable" target="_blank">Sustainable Development Goals</a> page, we are happy that the development of our project contributes to <span id="highlight">sustainable production and consumption systems</span>. The premise of our project involves <span id="highlight">generating wheat for human consumption in a manner that produces less biomass loss</span>, therefore a more sustainably produced variety of wheat than one that would make poorer use of resources through increased biomass loss. Even though our project itself contributes to this SDG, a significant amount of effort towards reaching the SDGs is increasing education around them to encourage others to pursue them as well. As such, we wanted to raise awareness of admirable strategies individuals have implemented to address this SDG, and inspire others to do the same. For this reason, we <span id="highlight">launched our ongoing podcast: "Sustainable Development in Synthetic Biology", launching three episodes with representatives from both industry and academia</span> working on varying approaches to reach this SDG. We acknowledge that promoting this podcast within Canada might mostly reach already-educated individuals that are aware of the SDGs, while our goal is to increase awareness of what activities can be done to reach the SDGs to individuals who are less aware of them. As such, we have been actively encouraging the <span id="highlight">podcast's promotion across four continents through sharing it with the network created from our International Accessibility Panel</span> (details below).</p>
+ <br>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/podcast.png" width="80%">
+ <figcaption>Podcast Links: RSS (<a href="https://rss.com/podcasts/ubcigem/" target="_blank">https://rss.com/podcasts/ubcigem/</a>) and <a href="https://open.spotify.com/show/0XoxgxlAEtypmucLuxUzig?si=f26944e25a004c89&nd=1" target="_blank">Spotify</a></figcaption>
+ </figure>
+ <br>
+ <h2 id="EndUserGoals">End-User Goals</h2>
+ <hr>
+ <p>Apart from the goals we had as product developers on the product features we wanted to provide to our consumers, the end-users needed to be ready to accept this innovation for our desired goals as product developers to be fulfilled. Our product will be able to ensure food security and support sustainable production systems, only if our end-users consume it appropriately and evade misuse of our technology. To ensure this, we wanted to <span id="highlight">strengthen GMO acceptability as well as the accessibility of synthetic biology innovations like ours across the world</span>. In order to gauge how our product may be perceived by consumers, we explored the discourse around the <span id="highlight">ethics of genetically modified crops and the acceptance of similar synthetic biology innovations globally</span>.</p>
+ <br>
+ <h5><b><i>GMO Acceptance:</i></b></h5>
+ <br>
+ <h5><b><u>"Let's Talk GMO" Panel</u></b></h5>
+ <p>Though the scientific aspects of our project seem cut and clear in order to increase sustainability by increasing heat tolerance in wheat, we understand that if this technology is not accepted by the public, it serves minimal purpose. The society plays an imperative role in any form of research undertaken as they are the end users of the product. Lack of public acceptance results in the inability to use the developed technology which thereby serves no purpose. As such, we wanted to dig deeper into <span id="highlight">understanding the societal, ethical, and social implications of gene editing</span> as well as <span id="highlight">understanding the public perceptions of genetically modified organisms</span>. The aim of this <span id="highlight">virtual panel</span> was to educate the general public regarding the <span id="highlight">benefits and drawbacks of GMO foods</span> via a meaningful discussion from a <span id="highlight">group of experienced panelists</span> ranging from social scientists and philosophers to corporate professionals.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/gmo-panel-1.png" alt="UBC Vancouver iGEM 'Lets Talk GMO' Panel" width="80%">
+ </figure>
+ <br>
+ <p>Our <span id="highlight">audience</span> consisted of a mixture of people ranging from students belonging to the iGEM community to those who had never heard of synthetic biology before. Our team guided the discussion by asking the panelists questions about various GMO-related topics but also let the conversation grow naturally as the panelists discussed and debated amongst themselves. There were opposing arguments in terms of the <span id="highlight">use and safety of GMOs</span> in a broader societal context. There was a general consensus that <span id="highlight">concerns surrounding GMOs</span> were at an all time low; however, simply weighing in the benefits along with the risks is not valid the way it may be in medicine. The goal of educating people should be based on a more bottom-up approach in the modern era rather than top-down. We need to find a way where the scientists' voices are heard more as they are the most trusted amongst the public as opposed to the government. The panel and question period were <a href="https://drive.google.com/drive/folders/1vh_rPBiNK_vTeF8Z1tSaKCfcpcD9f462?usp=sharing" target="_blank">recorded</a> and distributed to everyone who attended so they could also share it and generate further discussion.</p>
+ <br>
+ <h5><b><u>The C.O.D.E. Initiative Foundation</u></b></h5>
+ <p>Our two panel discussions brought to light the importance of a correct understanding of synthetic biology and how it can be utilized to tackle problems in an environmental or medical setting. Our team had the opportunity of providing learning resources to children and youth to learn more about GMO perspectives by starting a <span id="highlight">biology and genetics-based workshop series</span> in partnership with The C.O.D.E. Initiative Foundation called “<a href="https://www.thecodeinitiative.ca/classes/bits-of-bio-science-of-life" target="_blank">Bits of Bio: The Science of Life</a>â€.</p>
+ <br>
+ <p><a href="https://www.thecodeinitiative.ca/" target="_blank">The C.O.D.E Initiative Foundation</a> is a Vancouver-based, non-profit organization that provides affordable one-on-one and group, virtual and in-person, workshops for both neurodivergent and neurotypical students on various topics in STEM. Although the organization already offers many different courses on coding and robotics, there was a gap in topics geared towards the <span id="highlight">life sciences</span>. We <span id="highlight">developed and taught</span> an engaging module introducing the basic concepts of biology, providing students with an interesting and strong foundational introduction to the topic. As misconceptions about synthetic biology often stem from misinformation, our goal is to create an opportunity for youth to gain a holistic introduction to biology. We publicly share access to <a href="https://drive.google.com/drive/folders/1HtjVOZSd6lpYXFqweddheTTXzfeAoKys?usp=sharing" target="_blank">our teaching materials and curriculum</a>, which we used to successfully pilot our virtual, 1:1 workshops with several neurodivergent students.</p>
+ <br>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/bits-of-bio-info.png" alt="Bits of Bio: The Science of Life" width="80%" style="border: solid 2px var(--dark-color);">
+ <figcaption>Our "Bits of Bio: The Science of Life" Workshop Info as Advertised on The C.O.D.E. Initiative Foundation Website</figcaption>
+ </figure>
+ <br><br>
+ <h5><b><u>The Melius Mentorship Network</u></b></h5>
+ <p>To further <span id="highlight">extend our public outreach and to advocate for the accessibility of STEM for everyone</span>, our team collaborated with a youth-run organization to empower people of <span id="highlight">all educational and ethnic backgrounds</span> while exposing them to an innovative world of science and technology opportunities.</p>
+ <br>
+ <p>The <a href="https://www.meliusmentorship.com/" target="_blank">Melius Mentorship Network</a> is a student-led, non-profit organization at the University of British Columbia (UBC), which aims to provide personalized mentorship and educational workshops for refugee youth and newcomers to Canada. Our team carried out a <span id="highlight">hands-on, biology-based workshop to high school immigrants and refugees</span>, educating them about the utility of synthetic biology research and providing a look into the molecular world of DNA and its modifications. We facilitated a <span id="highlight">DNA extraction activity and a gene editing activity</span>, where they had to solve real-world problems through designing organisms with novel features. We included examples from the real world, such as the production of Golden Rice. Given the nature of their status in Canada, the hands-on activities were especially appreciated due to language barriers. The seven students demonstrated enthusiasm and admiration for the field of biological engineering after the session, inquiring about paths one can take to incorporate this into their career.</p>
+ <br>
+ <figure>
+ <div class="row">
+ <img class="col-6" src="https://static.igem.wiki/teams/4296/wiki/images/56.jpg" alt="Strawberry DNA Extraction Activity with The Melius Mentorship Network">
+ <img class="col-6" src="https://static.igem.wiki/teams/4296/wiki/images/65.jpg" alt="Strawberry DNA Extraction Activity with The Melius Mentorship Network">
+ <figcaption>Hands-on Strawberry DNA Extraction Activity at our Workshop with The Melius Mentorship Network</figcaption>
+ </div>
+ </figure>
+ <br>
+ <h5><b><i>Access to Synthetic Biology Innovations:</i></b></h5>
+ <br>
+ <h5><b><u>"International Accessibility to Synthetic Biology Innovations" Panel</u></b></h5>
+ <p>Inspired by our discussion with professionals working in the biotechnology sector and academic researchers from around the world throughout the project, we wanted to gain further insight on the <span id="highlight">barriers to accessing biotechnology innovations across the globe</span>. If our technology becomes inaccessible to certain regions of the world, it will not reach certain target audiences that are affected by the issue we are trying to address. It was highlighted in our GMO panel that an individual’s educational background and their environment are major contributors to their opinion on the use of synthetic biology as a solution to the climate crisis and its subsequent effects such as food insecurity. The goal was to learn about the obstacles professionals from various countries face in the realm of synthetic biology, and contrast the network and environment support between different countries to understand how developing countries can improve this ecosystem. Ultimately, we wanted our technology to reach all corners of the globe regardless of accessibility issues related to GMO development and use. To reach this goal, we created a <span id="highlight">panel discussing the differences in GMO use in different countries</span> in collaboration with the <a href="https://2022.igem.wiki/iiser-pune-india/" target="_blank">IISER Pune iGEM</a> team from India.</p>
+ <br>
+ <p><span id="highlight">Speakers from South America, North America, Asia, and Africa at different stages in their professional career</span> were invited to answer questions and provide insight on their experiences. When discussing the <span id="highlight">legal framework for biotechnology in each country</span>, there was a common theme among the speakers from <span id="highlight">Uganda, Peru, and India</span> that a clear set of laws and regulations for synthetic biology had yet to be set, which acts as a barrier when seeking approval of novel projects. The large disparity in the <span id="highlight">accessibility to equipment and funding</span> in each country was also brought to light, from Uganda, where there are little to no synthetic biology education opportunities, to Canada, where there is a vast number of opportunities that lie within academia and industry. In most speakers’ experiences, spreading awareness about synthetic biology was exclusively carried out by students, start-up companies, and community labs. It became evident that there is a <span id="highlight">need for a standardized regulatory system and government support</span> to allow for the growth of sub-biotechnology sectors in developing countries.</p>
+ <br>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/international-panel.png" alt="International Accessibility to SynBio Innovations Panel" width="80%">
+ <figcaption>UBG Vancouver iGEM’s "International Accessibility to Synthetic Biology" Panel</figcaption>
+ </figure>
+ <br><br>
+ <div class="accordion" id="accordionExample">
+ <div class="accordion-item">
+ <h2 class="accordion-header" id="references">
+ <button class="accordion-button collapsed" type="button" data-bs-toggle="collapse" data-bs-target="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
+ References
+ </button>
+ </h2>
+ <div id="collapseOne" class="accordion-collapse collapse" aria-labelledby="references" data-bs-parent="#accordionExample">
+ <div class="accordion-body">
+ <p><span id='highlight'>[1]</span> Chugh, A. (2021). How can technological advancements in synthetic biology benefit everyone? An expert explains. <i>World Economic Forum.</i> <a href="https://www.weforum.org/agenda/2021/11/synthetic-biology-can-benefit-all-expert-explains-how/" target="_blank">https://www.weforum.org/agenda/2021/11/synthetic-biology-can-benefit-all-expert-explains-how/</a></p>
+ <p><span id='highlight'>[2]</span> Shisanya, S.O. & Hendriks, S.L. (2011). The contribution of community gardens to food security in the Maphephetheni uplands, determined by the Household Food Insecurity Access Scale. <i>Development Southern Africa, 28(4), 509-526.</i></p>
+ </div>
+ </div>
+ </div>
+ </div>
+ </div>
+ </div>
+ </body>
+</html>
+{% endblock %}
\ No newline at end of file
diff --git a/wiki/pages/model.html b/wiki/pages/model.html
index 007e1328883175975d74604703ed0fd8f8497f21..ca609a69cad0f7b7b72c510eecbc41bad675f8f5 100644
--- a/wiki/pages/model.html
+++ b/wiki/pages/model.html
@@ -47,7 +47,7 @@
<ol>
<li>To confirm the rationale behind our gene circuit designs, we performed <span id="highlight">RNA-seq analysis</span> to determine differentially expressed genes and differentially regulated pathways in heat and drought-stressed <i>T. aestivum</i> wheat.</li><br>
<li>To better understand our genetically engineered system, we developed <span id="highlight">mathematical models for predicting heat-inducible gene expression and enzyme kinetics</span> for our reactions of interest.</li><br>
- <li>To increase the stability of our enzymes under heat stress, we performed preliminary <span id="highlight">molecular modelling using PyMOL and PyRosetta</span> to identify active site mutations that would lead to optimal enzyme thermostability.</li><br>
+ <li>To increase the stability of our enzymes under heat stress, we performed preliminary <span id="highlight">molecular modelling using PyMOL and PyRosetta</span> to identify active site mutations that would lead to optimal enzyme thermodynamic stability.</li><br>
<li>To aid the proposed implementation of our project in the field, we designed and tested a portable <span id="highlight">Arduino-based fluorometer device</span> to detect heat stress in the wheat through fluorescence activation of our engineered gene circuits.</li>
</ol>
</p>
@@ -69,16 +69,48 @@
<br><br>
<figure>
<img src="https://static.igem.wiki/teams/4296/wiki/images/wheat-expression-browser-sbpase.png" alt="Example Use of the Wheat Expression Browser Tool" width="100%">
- <figcaption>Comparing the Average Expression Levels of the SBPase Gene Homoeologs Across Different Sample Conditions</figcaption>
+ <figcaption>Example Use Case: Comparing the Average Expression Levels of the SBPase Gene Homoeologs Across Different Sample Conditions</figcaption>
</figure>
<br>
- <p><b><u>Exploratory Analysis:</u></b></p>
- <p>We first performed a comparative analysis of the transcriptome profiles of the wheat seedling leaves under drought stress, heat stress, and combined stress conditions using a normalized (TPM - Transcripts Per Million) expression count matrix.</p>
+ <p><b><u>Comparative Analysis:</u></b></p>
+ <p>To better understand and compare the different molecular effects of heat vs. drought stress in wheat, we performed a comparative analysis of the transcriptome profiles of the wheat seedling leaves under drought stress, heat stress, and combined stress conditions using a normalized (TPM - Transcripts Per Million) expression count matrix.</p>
<figure>
- <img src="https://static.igem.wiki/teams/4296/wiki/images/2d-pca.jpg" alt="2D PCA Plot of Samples" width="75%">
- <figcaption>Principal Component Analysis (PCA) with the Proportion of Variance Explained by Each PC</figcaption>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/2d-pca.jpg" alt="2D PCA Plot of Samples" width="55%">
+ <figcaption>2D Principal Component Analysis (PCA) with the Proportion of Variance Explained by Each PC</figcaption>
+ <br>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/3d-pca.png" alt="3D PCA Plot of Samples" width="45%" style="border: none;">
+ <figcaption>3D Principal Component Analysis (PCA) with the Proportion of Variance Explained by Each PC</figcaption>
+ </figure>
+ <br>
+ <p>In the dimensionality reduction plots above, <span id="highlight">the more similar datapoints (ie. samples) are in terms of their gene expression profiles, the closer they are located in space</span>. From these PCA plots, we observe very high similiarity between the biological replicates as expected, and also note these four distinct groups of sample conditions clustered together based on similarity: heat and combined stress (1h treatment), heat and combined stress (6h treatment), control (untreated) and drought stress (1h treatment), and drought stress (6h treatment).</p>
+ <p>These results suggest that <span id="highlight">the mRNA (ie. gene expression) profiles of drought-stressed wheat differ significantly from that of heat or combined stress</span>, and they show complex relationships depending on the length of stress treatment (as seen by the distinctly separate 1h vs. 6h drought stress samples).</p>
+
+ <p><b><u>Differential Gene Expression Analysis:</u></b></p>
+ <p>Next, we performed statistical testing between the gene expression counts of different sample conditions in order to determine <span id="highlight">significant differentially expressed genes</span>. As intense heat and drought conditions often coincide in the real world, as was pointed out by several agricultural experts and farmers we spoke to for our <a href="https://2022.igem.wiki/ubc-vancouver/human-practices" target="_blank">Integrated Human Practices</a> work, we mainly focused on comparing the combined (heat and drought), long-term (6h treatment) stress condition to the untreated control condition.</p>
+ <p>Using the <a href="https://bioconductor.org/packages/release/bioc/html/edgeR.html" target="_blank">edgeR</a> software package, we identified 2079 differentially expressed genes (890 upregulated and 1189 downregulated) in wheat seedling leaves in the long-term combined stress condition (HD6h) compared to the control (using <span id="highlight">extremely stringent filtering</span> conditions of fold change ≥ 32 and false discovery rate (FDR)-adjusted P <= 0.001).</p>
+ <br>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/interactive-volcano-plot.png" width="100%" style="border: none;">
+ <figcaption>Screenshot of an interactive volcano plot where each datapoint represents a gene. On hover, the gene name is displayed.</figcaption>
+ <br>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/example-use.png" width="60%">
+ <figcaption>As an example and sanity check, the highly upregulated gene from the plot above was searched and revealed to be a heat shock chaperone protein.</figcaption>
</figure>
+ <br>
+ <p><b><u>Gene Ontology (GO) and Plant Reactome Analysis:</u></b></p>
+ <p>To functionally characterize the differentially expressed genes and understand their effect on the wheat plant in a biological context, we used the <a href="https://bioconductor.org/packages/release/bioc/html/biomaRt.html" target="_blank">biomaRt</a> software package to look at the <span id="highlight">top (ie. most frequent) Gene Ontology terms</span> associated with the upregulated or downregulated genes, as well as look at their <span id="highlight">Plant Reactome reactions and pathways</span>.</p>
+ <br>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/upreg.png" width="100%" style="border: none;">
+ <figcaption>The top Molecular Functions, Biological Processes, and Cellular Components associated with the upregulated genes in the HD6h condition. We observe many terms associated with <span id="highlight">heat shock and chaperone proteins</span> (ex: "response to heat", "protein folding", "unfolded protein binding"), <span id="highlight">regulation of gene expression</span> (ex: "regulation of transcription, DNA-templated", "DNA-binding transcription factor activity"), and <span id="highlight">osmolarity</span> (ex: "response to salt stress").</figcaption>
+ <br><br>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/downreg.png" width="100%" style="border: none;">
+ <figcaption></figcaption>
+ <br><br>
+ </figure>
+
+ <p>For our complete analysis and scripts, please refer to our centralized <a href="https://github.com/UBC-iGEM/wheat_RNAseq_analysis" target="_blank">GitHub repository</a>.</p>
<br><br>
<!-- MATHEMATICAL MODELLING -->
@@ -111,7 +143,7 @@
<figcaption>Expression profile of the system’s promoter, transcription factor, and promoter-transcription factor complex. The promoter and transcription factor plotlines are overlaid.</figcaption>
</figure>
<br>
- <p>Our attempt at finding a K<sub>d</sub> value for the transcription factor through literature review was unsuccessful. It is also quite difficult and expensive to determine through wet-lab techniques, and the team was unable to find software with sufficient accuracy to predict the dissociation constant values. Hence, the K<sub>d</sub> value from a homologous protein was used in our model instead<span id="highlight"><sup>[TODO]</sup></span>. This choice is justified by the relatively low dependence of the [PTF] on the K<sub>d</sub> value, as visualized by a SimBiology Global Variance Analysis simulation, presented below:</p>
+ <p>Our attempt at finding a K<sub>d</sub> value for the transcription factor through literature review was unsuccessful. It is also quite difficult and expensive to determine through wet-lab techniques, and the team was unable to find software with sufficient accuracy to predict the dissociation constant values. Hence, the K<sub>d</sub> value from a homologous protein was used in our model instead. This choice is justified by the relatively low dependence of the [PTF] on the K<sub>d</sub> value, as visualized by a SimBiology Global Variance Analysis simulation, presented below:</p>
<figure>
<img src="https://static.igem.wiki/teams/4296/wiki/drylab/image11.png" alt="Global Variance Analysis of the Transcription Factor Model" width="70%">
<figcaption>Global Variance Analysis of the Transcription Factor Model</figcaption>
@@ -154,6 +186,13 @@
<img src="https://static.igem.wiki/teams/4296/wiki/drylab/image33.png" alt="SBPase Production and Calvin Cycle Pathway" width="80%">
</figure>
<br>
+ <p>We also conducted global variance analysis to visualize the potential effects the synthetic pathway would have on the starch production of the imported Calvin-Benson Cycle, and presented it below:</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/drylab/cbbmodified-gsa.png" width="50%">
+ <figcaption>Global Sensitivity Analysis Demonstrating the Effect of Transcription Factor, Promoter, and SBPase concentrations on Starch Production</figcaption>
+ </figure>
+ <br>
+
<p><b><u>Modeling the ACCD-Ethylene Pathway:</u></b></p>
<p>Once again, we reuse the transcription factor (Hill Kinetics) portion of the derivation to generate the same equation for the promoter-transcription factor complex. Furthermore, since we continue to use the Briggs-Haldane derivation of Michaelis-Menten kinetics, we obtain the same expression for the V<sub>max</sub> of our new enzyme system, with different species:</p>
<figure>
@@ -185,6 +224,7 @@
<p>As MATLAB SimBiology did not have a pre-built multi-substrate kinetics function, it was manually encoded as an "Undefined reaction". Modeling this system in SimBiology yields the following expression profile:</p>
<figure>
<img src="https://static.igem.wiki/teams/4296/wiki/drylab/image32.png" alt="Expression Profile of the ACCD System" width="80%">
+ <figcaption>Dimensionless Expression Profile of Molecules Against Time</figcaption>
</figure>
<br>
<p>Following this, the team conducted sensitivity analysis of the expression system, measuring the sensitivity of ethylene concentration against varying reaction rates and species. This produced the following Sobol plots:</p>
@@ -194,6 +234,19 @@
</figure>
<br>
<p>Once again, we see that none of the reaction rates have a very strong direct effect on ethylene production, except the Michaelis-Menten constants for ACC deaminase and ACC oxidase. It is reasonable for the K<sub>M<sub>b</sub></sub> value (Michaelis-Menten constant for ACC oxidase) to score higher as it is the governing reaction rate of ethylene production. Among individual species, it is justified that the highest scoring species are ACC (the precursor molecule to ethylene) and ACC oxidase (enzyme responsible for ethene production).</p>
+
+ <p><b><u>Conclusion:</u></b></p>
+ <p>Through our mathematical modelling work, the team was able to gain meaningful insight into our <span id="highlight">gene circuits' enzyme kinetics</span> and <span id="highlight">identify the most important parameters affecting each of the simulated models</span>. Furthermore, the team demonstrated the SBPase circuit’s viability by connecting it to the published .SBML model of the Calvin Benson Cycle as a proof of concept.</p>
+ <p>Our work can be furthered by an exploration of the effects of ACC deaminase production on the Yang Cycle, which is analogous to the work conducted with the Calvin-Benson Cycle. However, the team was unable to find a working model of the Yang Cycle from literature, and did not have the time to calculate parameters required to create a model from scratch.</p>
+ <p>Mathematical analysis can also be applied to further characterize the operating range of our system, by measuring changes to enzyme expression (using fluorescence output as a proxy) as a function of temperature. Although UBC iGEM attempted to do so with their current experimental data, we were not able to collect a sufficient number of tested datapoints over a large temperature range due to time restrictions. An incomplete quadratic function modelling the temperature inducibility of the promoter has been included below. Analysis was completed on <a href="https://www.vernier.com/product/logger-pro-3/" target="_blank">Logger Pro</a>.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/drylab/quadratic.png" width="70%">
+ <figcaption>A poorly fitting quadratic function. Additional temperatures need to be tested to increase resolution of the data.</figcaption>
+ <!-- <br>
+ <img src="https://static.igem.wiki/teams/4296/wiki/drylab/gaussian.png" width="70%">
+ <figcaption>The Gaussian function is predicting the ideal domain needed to fit a parabolic function. Future experiments should collect temperature readings between 20°C and 50°C</figcaption> -->
+ </figure>
+ <br>
<p>For full equation derivations, MATLAB Simbiology files, and further details, please refer to this central <a href="https://github.com/UBC-iGEM/mathematical-modelling-2022" target="_blank">GitHub repository</a> where we have uploaded all of our mathematical modelling work and documentation to allow for exploration and reproducibility.</p>
<br><br>
@@ -304,7 +357,7 @@
</p>
<p>However, this workflow was realized to be computationally expensive and time-consuming, so our team was only able to compute docking energies for interactions between the TaHsp70d promoter and TaHsfA2b transcription factor. A PDB structure of the TaHsp70d promoter sequence was created in PyMOL using a script provided from the Supercomputing Facility for Bioinformatics and Computational Biology<span id="highlight"><sup>[11]</sup></span>.</p>
<p>Mutations to the DNA promoter sequence were created manually on <a href="https://www.cgl.ucsf.edu/chimera/" target="_blank">UCSF Chimera</a> using the ‘Rotamer’ function, which would predict the most probable conformation of the target mutation. This process was repeated 10 times to create 10 mutated PDB structures of the promoter.</p>
- <p>Lastly, these structures were docked using <a href="http://hdock.phys.hust.edu.cn/" target="_blank"></a>, a web tool specializing in calculating Protein-DNA docking scores. A table of corresponding docking scores is presented below:</p>
+ <p>Lastly, these structures were docked using <a href="http://hdock.phys.hust.edu.cn/" target="_blank"></a>, a web tool specializing in calculating Protein-DNA docking scores. <span id="highlight">A more negative docking score means a more possible binding model</span>, but the score should not be treated as the absolute true binding affinity of two molecules because it has not been calibrated to the experimental data. A table of corresponding docking scores is presented below:</p>
<figure>
<img src="https://static.igem.wiki/teams/4296/wiki/drylab/docking-score-table.png" alt="Hdock Results" width="80%" style="border: none;">
<figcaption>Protein-DNA Docking Score Results From Hdock</figcaption>
@@ -484,8 +537,13 @@
<span class="visually-hidden">Next</span>
</button>
</div>
- <br><br>
-
+ <br>
+ <p><b><u>Conclusion:</u></b></p>
+ <p>With the <span id="highlight">circuit soldered together and fitted to our 3D printed prototype</span>, we hope that the device can serve as a <span id="highlight">portable and low-cost plate reader for wheat diagnostics into the field at the site of heat impact</span>. As high temperatures activate our promoter, the GFP (or other fluorescent proteins) in our construct are expressed to indicate gene activation. With this construct in transgenic wheat, the fluorometer device would then be used in the field to check wheat crops for heat stress without the need to bring plant samples into a lab. This provides independent confirmation to farmers with their transgenic wheat.</p>
+ <p>However, the current prototype is far from perfect. There is still a need for the addition of a soft material to block out ambient light as the device clamps over a leaf as per our original concept design. The current prototype also connects to a laptop for power and outputs data there as well. The final design will be battery-powered and provide confirmation of construct activity with LED status lights and displays. Additionally, the device would also incorporate readings from other sensors like that of temperature and humidity. The GFP device would provide on-site support for monitoring the transgenic, heat-tolerant wheat.</p>
+ <p>For further details such as the complete list of parts and costs, as well as full documentation of our entire workflow, please refer to our centralized <a href="https://github.com/UBC-iGEM/hardware-2022" target="_blank">GitHub repository</a>.</p>
+ <br>
+
<!-- REFERENCES -->
<div class="accordion" id="accordionExample">
<div class="accordion-item">
diff --git a/wiki/pages/partnership.html b/wiki/pages/partnership.html
index 10730bc63e7188a47f8f8a2e3a8c1fcac74cd5fa..7502a4d59f9e6024a017915d7feb43f357fe0e1f 100644
--- a/wiki/pages/partnership.html
+++ b/wiki/pages/partnership.html
@@ -12,6 +12,14 @@
display: block;
margin: auto;
}
+
+ iframe {
+ display: block;
+ margin: auto;
+ width: 80%;
+ height: 750px;
+ box-shadow: 1px 1px 5px 5px #f0742d8e;
+ }
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</head>
<body>
@@ -36,7 +44,23 @@
<br><br>
<h2 id="DryLab">Dry-Lab</h2>
<hr>
- <p>Starting in July, <span id="highlight">our teams exchanged expertise on reaction kinetics modeling and climate modeling</span>.<br><br>Our team’s <a href="https://2022.igem.wiki/ubc-vancouver/model" target="_blank">mathematical modeling</a> work on modeling factors of the synthesis of ethylene inspired the IISER Pune team to do the same for their ACCD reaction. Additionally, they were interested in measuring the levels of ACCD released by plant roots for its uptake by bacteria like the ones they were engineering. They asked us to design and execute Wet-Lab experiments for these measurements. Due to time and resource constraints, we were unable to carry out the measurement experiments, and thus only planned them.<br><br>On the other hand, their team was implementing <span id="highlight">climate modeling</span> for predicting and measuring the land areas of the world that are affected by waterlogging. We believed our project could benefit from a similar approach to view the geographic areas whose wheat yield would be affected by heat. Therefore, they offered to model the parameters we were interested in obtaining from <span id="highlight">satellite data</span> (ex: areas that are growing spring wheat, precipitation, temperature during the grain filling season) to then overlap for a summary of locations that would be affected by decreased wheat yield. They were able to obtain information and begin the modeling for a few of the parameters, but not synthesize it all into a single model. Regardless, we believe our collaboration allowed both our teams to gain further insight into our project, especially past the competition if these efforts are sustained.</p>
+ <p>Starting in July, <span id="highlight">our teams exchanged expertise on reaction kinetics modeling and climate modeling</span>.<br><br>Our team’s <a href="https://2022.igem.wiki/ubc-vancouver/model" target="_blank">mathematical modeling</a> work on modeling factors of the synthesis of ethylene inspired the IISER Pune team to do the same for their ACCD reaction. Additionally, they were interested in measuring the levels of ACCD released by plant roots for its uptake by bacteria like the ones they were engineering. They asked us to design and execute Wet-Lab experiments for these measurements. Due to time and resource constraints, we were unable to carry out the measurement experiments, and thus only planned them, as detailed below.</p>
+ <div class="accordion" id="accordionExample">
+ <div class="accordion-item">
+ <h2 class="accordion-header" id="experiments">
+ <button class="accordion-button collapsed" type="button" data-bs-toggle="collapse" data-bs-target="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
+ Wet-Lab Experimental Design & Plans
+ </button>
+ </h2>
+ <div id="collapseOne" class="accordion-collapse collapse" aria-labelledby="experiments" data-bs-parent="#accordionExample">
+ <div class="accordion-body">
+ <iframe src="https://static.igem.wiki/teams/4296/wiki/pdf/iiser-pune-partnership-wet-lab-design.pdf"></iframe>
+ </div>
+ </div>
+ </div>
+ </div>
+ <br>
+ <p>On the other hand, their team was implementing <span id="highlight">climate modeling</span> for predicting and measuring the land areas of the world that are affected by waterlogging. We believed our project could benefit from a similar approach to view the geographic areas whose wheat yield would be affected by heat. Therefore, they offered to model the parameters we were interested in obtaining from <span id="highlight">satellite data</span> (ex: areas that are growing spring wheat, precipitation, temperature during the grain filling season) to then overlap for a summary of locations that would be affected by decreased wheat yield. They were able to obtain information and begin the modeling for a few of the parameters, but not synthesize it all into a single model. Regardless, we believe our collaboration allowed both our teams to gain further insight into our project, especially past the competition if these efforts are sustained.</p>
<figure>
<img src="https://static.igem.wiki/teams/4296/wiki/images/iiser-pune.png" alt="Zoom meeting with IISER-Pune">
<figcaption>UBC Vancouver iGEM and IISER Pune iGEM Team Meeting</figcaption>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index b39294e6024f917e114568cfe661eaf8a83904c9..5129e687fa22085fb5f62ba8d667dfa8ad381422 100644
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+++ b/wiki/pages/results.html
@@ -126,8 +126,12 @@
<figcaption>GFP fluorescence levels of protoplasts transfected with SBPase-iRFP, ACCD-BFP, or constitutively expressed GFP.</figcaption>
</figure>
<br>
- <p>We wanted to investigate how much of the lack of fluorescent data could be explained by protoplasts that lost viability during the heat shock; therefore, <span id="highlight">we measured viability before and after heat shock</span>. Previous studies<span id="highlight"><sup>[3]</sup></span> showed no viability of protoplasts at 55°C, so we wanted to explore if this was also the case at lower temperatures. <span id="highlight">As shown below, protoplast viability did not vary considerably between temperatures, with 45°C showing the most decreased viability</span>, potentially explaining the decreased fluorescence data seen at this temperature. Fluorescence did not decrease to the degree of protoplasts losing viability at this temperature; thus we hypothesize that our fluorescent proteins are remaining inside non-viable cells for a period of time before they are completely degraded. This means that some of the fluorescense can be attributed to non-viable protoplasts.</p>
- <p><span id="highlight">GFP fluorescence was also observable by fluorescence microscopy</span>. The morphology of protoplasts is more irregular than the consistent spherical shape observed during protoplast purification and isolation. This change is likely due to handling and pipetting the protoplasts repeatedly, shearing them and modifying their shape. Some of these morphological extremes are merely pieces of protoplasts that are still expressing GFP: this supports our theory that non-viable or sheared protoplasts are still expressing fluorescent proteins for a period of time before their degradation, affecting fluorescence values.</p>
+ <p>We wanted to investigate how much of the lack of fluorescent data could be explained by protoplasts that lost viability during the heat shock; therefore, <span id="highlight">we measured viability before and after heat shock</span>. This was done through adding 1 µL of 1% Evans Blue Dye to 25 µL of protoplasts and counting viable (non-blue) protoplasts on the hemocytometer. Viability percentage was calculated by the number of viable protoplasts over total. Previous studies<span id="highlight"><sup>[3]</sup></span> showed no viability of protoplasts at 55°C, so we wanted to explore if this was also the case at lower temperatures. <span id="highlight">As shown below, protoplast viability did not vary considerably between temperatures, with 45°C showing the most decreased viability</span>, potentially explaining the decreased fluorescence data seen at this temperature. Fluorescence did not decrease to the degree of protoplasts losing viability completely at this temperature; thus we hypothesize that our fluorescent proteins are remaining inside non-viable cells for a period of time before they are completely degraded. This means that some of the fluorescense can be attributed to non-viable protoplasts.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/viability-temperature-treatments.png" width="80%">
+ </figure>
+ <br>
+ <p><span id="highlight">GFP fluorescence was also observable by fluorescence microscopy</span>. The morphology of protoplasts is more irregular than the consistent spherical shape observed during protoplast purification and isolation. This change is likely due to handling and pipetting the protoplasts repeatedly, shearing them and modifying their shape. Some of these morphological extremes are merely pieces of protoplasts that are still expressing GFP: this supports our theory that non-viable or sheared protoplasts are still expressing fluorescent proteins for a period of time before their complete degradation, affecting fluorescence values.</p>
<figure>
<img src="https://static.igem.wiki/teams/4296/wiki/images/221002-gfp-protplasts-24h-post-tx-rt-17.jpeg" width="75%" alt="Fluorescence Micrograph of GFP-expressing Protoplasts">
<figcaption>Wheat protoplasts transfected with GFP 24 hours after transfection, 20x magnification.</figcaption>
@@ -146,22 +150,22 @@
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- <img src="https://static.igem.wiki/teams/4296/wiki/images/protoplasts-cell-wall-enzyme.jpg" class="d-block w-25" alt="Plant Cell Wall Enzyme Digest">
+ <div class="carousel-item active" data-bs-interval="100000">
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/protoplasts-cell-wall-enzyme.jpg" class="d-block w-50" alt="Plant Cell Wall Enzyme Digest">
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<br><br>
<p>Washing cut plant leaves in cell wall enzyme solution.</p>
</div>
</div>
- <div class="carousel-item" data-bs-interval="10000">
- <img src="https://static.igem.wiki/teams/4296/wiki/images/protoplasts-w5.jpg" class="d-block w-25" alt="W5 Media">
+ <div class="carousel-item" data-bs-interval="100000">
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/protoplasts-w5.jpg" class="d-block w-50" alt="W5 Media">
<div class="carousel-caption d-none d-md-block">
<br><br>
<p>Washing protoplasts with W5 media.</p>
</div>
</div>
<div class="carousel-item">
- <img src="https://static.igem.wiki/teams/4296/wiki/images/protoplasts-microscopy.png" class="d-block w-25" alt="Protoplasts">
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/protoplasts-microscopy.png" class="d-block w-50" alt="Protoplasts">
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<br><br>
<p>Isolated protoplasts.</p>
@@ -212,11 +216,16 @@
<figcaption><span id="highlight">Figure 2</span> - Stained, non-viable protoplasts permeable to Evan's blue dye observed.</figcaption>
</figure>
<figure>
- <img src="" alt="Lysed Protoplasts" width="55%">
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/lysed-protoplasts.png" alt="Lysed Protoplasts" width="35%" style="border: 2px solid var(--dark-color);">
<figcaption><span id="highlight">Figure 3</span> - Unstained but lysed protoplasts observed. The small clusters indicate chloroplasts.</figcaption>
</figure>
<br>
+ <p><b><u>Storage Condition Optimization</u></b></p>
<p><span id="highlight">As no long-term storage options currently exist for wheat protoplasts in the literature, we tested our own storage method</span> based off a paper where <i>F. brasiliense</i> protoplasts were stored frozen at − 80°C for up to two years and still showed viability<span id="highlight">[5]</span>. Being able to store protoplasts would be very useful because obtaining these systems is time consuming and not always reliably guaranteed. Finding a way to store wheat protoplasts allows us to use them in the future without worrying about yield and the possibility of not obtaining sufficient amounts for downstream experiments. We attempted to store our protoplasts by adding DMSO to 7% total concentration 2 mL cryotubes and cooling them in the -80°C freezer. The concentration of protoplasts frozen was twice the concentration (2.5 x 10<sup>6</sup>) needed for transfection, as we expected ~50% to lose viability during freezing or thawing. Protoplasts were thawed on ice, pelleted, and the supernatant removed. Subsequent washes were done with W5 to remove DMSO, and the protoplasts were finally resuspended in MMG. When the protoplasts were checked for viability using the hemocytometer the next day, we discovered that almost all of them were dead. We hypothesized that the protoplasts were killed by flash-freezing since we had no method to control the rate of temperature change after putting them in the freezer. Given further time and resources, we could potentially have improved the efficiency of this storage method by purchasing a Thermo Scientificâ„¢ Mr. Frostyâ„¢ Freezing Container, which controls the rate of cooling to approximately -1 °C/min.</p>
+ <p>After this initial testing at -80°C, we hypothesized that storage at higher temperatures could increase viability after storage in DMSO. We used the SBPase transfection condition as our sample to test longer-term viability testing. The graph below includes the temperatures before treatment, after 2-hour treatment, and viability after 24-hour storage in room temperature, 4°C and -80°C. Viability remained nearly the same after 24 hours and 48 hours at all conditions. The 4°C treatment after 24 hours showed an increased amount of viability, which was likely a bias error from counting on the hemocytometer.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/4296/wiki/images/viability-after-storage.png" width="60%">
+ </figure>
<br>
<h2 id="FutureDirections">Future Directions</h2>
<hr>
@@ -252,11 +261,11 @@
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- <button class="accordion-button" type="button" data-bs-toggle="collapse" data-bs-target="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
+ <button class="accordion-button collapsed" type="button" data-bs-toggle="collapse" data-bs-target="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
References
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</h2>
- <div id="collapseOne" class="accordion-collapse collapse show" aria-labelledby="references" data-bs-parent="#accordionExample">
+ <div id="collapseOne" class="accordion-collapse collapse" aria-labelledby="references" data-bs-parent="#accordionExample">
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<p><span id='highlight'>[1]</span> Hu, Y., Song, D., Gao, L., Ajayo, B. S., Wang, Y., Huang, H., Zhang, J., Liu, H., Liu, Y., Yu, G., Liu, Y., Li, Y., & Huang, Y. (2020). Optimization of isolation and transfection conditions of maize endosperm protoplasts. <i>Plant Methods, 16(1).</i> <a href="https://doi.org/10.1186/s13007-020-00636-y" target="_blank">https://doi.org/10.1186/s13007-020-00636-y</a></p>
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