diff --git a/static/style.css b/static/style.css
index b8a898858384b3ea6012f3f60b1169af84c35618..ed4550b9d275dcc452b55a81349e6068e367606c 100644
--- a/static/style.css
+++ b/static/style.css
@@ -725,7 +725,7 @@ footer a:hover {
}
#index-empty {
- height: 950vh;
+ height: 775vh;
}
.model-page-img {
@@ -930,6 +930,13 @@ figure img {
cursor: pointer;
}
+.pdf-protocol {
+ display: flex;
+ width: 70vw;
+ height: 70vh;
+
+}
+
/*
Adapted from:
https://css-tricks.com/hexagons-and-beyond-flexible-responsive-grid-patterns-sans-media-queries/
diff --git a/wiki/pages/acknowledgements.html b/wiki/pages/acknowledgements.html
index 59cf475331ef0f8d0ff037863f249f8a9f7a88c2..ae28e1e2db453366f54f159bd0a47960da2a1093 100644
--- a/wiki/pages/acknowledgements.html
+++ b/wiki/pages/acknowledgements.html
@@ -1,29 +1,26 @@
{% extends "layout.html" %}
-
+
{% block title %}Acknowledgements{% endblock %}
-{% block lead %} Subtitle.{% endblock %}
+{% block lead %}{% endblock %}
{% block page_content %}
<div class="row mt-4">
<div class="col">
- <p>We would like to deeply thank our PIs, advisors and former team members for all the hard work invested in the project and confidence in our work. To all of you: thank you very much!</p>
+ <p>We would like to deeply thank our PIs, advisors and former team members for all the hard work invested in the project and confidence in our work. To all of you: thank you very much!</p>
- <p>We also need to thank all the researchers who gave their time and facilities so that we could carry out various stages of the Cellulopolis project:</p>
+ <p>We also need to thank all the researchers who gave their time and facilities so that we could carry out various stages of the Cellulopolis project:</p>
- <p>Prof. Hernane Barud’s team</p>
- <p> Prof. Hernandes Carvalho´s team</p>
- <p> Prof. Carmen Versissima´s team</p>
- <p> Prof. Marcelo Brocchi´s team</p>
- <p> Prof. André Damasio and Fernanda Lopes de Figueiredo</p>
- <p> Laboratory of Genomics and Bioenergy (LGE)</p>
+ <p>Prof. Hernane Barud’s team</p>
+ <p> Prof. Hernandes Carvalho´s team</p>
+ <p> Prof. Carmen Versissima´s team</p>
+ <p> Prof. Marcelo Brocchi´s team</p>
+ <p> Prof. André Damasio and Fernanda Lopes de Figueiredo</p>
+ <p> Laboratory of Genomics and Bioenergy (LGE)</p>
<p>Without you all this would have been nothing more than the brainchild of undergraduate students.</p>
<p>A special thanks to our advisor Giovana Makluf, always very helpful and critical in helping us to polish the project into the little jewel it is today.</p>
<p>And finally, a huge thank you to everyone who helped us in our funding campaigns or helped us in any other way. The DNA of this project carries the contribution of each and every one of you.</p>
-
-
- <p> Gratiluz! %#58158</p>
</div>
</div>
{% endblock %}
diff --git a/wiki/pages/attributions.html b/wiki/pages/attributions.html
index 233e7f0252da5b121cd9037c45db0fcbb5bd1233..7fc50e11ca7344a82a0e724f65b559218b3520f6 100644
--- a/wiki/pages/attributions.html
+++ b/wiki/pages/attributions.html
@@ -38,8 +38,7 @@
<h1>Principal Investigator and Advisors</h1>
<p><b>Elizabeth Bilsland</b> is a pivotal member of our team. As a PI, she provided laboratory training to team members, helped to secure sponsorship, dealt with red-tape involved in the import of iGEM parts and IDT custom DNA, performed E. coli transformations, minipreps and cloning, registered new parts, and helped with writing. Designed experiments, conceptualized the Bacterial Cellulose game, and provided overall support for the team. In addition, she was the main responsible for the data curation in our project so that it can be used in the future by other iGEM teams or members of the synthetic biology community.</p>
- <p><b>Fellipe da Silveira Bezerra de Mello</b> acted as team advisor in key stages of Cellulopolis. Provided fundamental lab and project support in the wet and dry lab experiments.</p>
- <p><b>Lucas Miguel de Carvalho</b> acted as team advisor in the validation of the genome-scale metabolic model and provide support in the dry lab experiments.</p>
+ <p><b>Lucas Miguel de Carvalho</b> acted as team advisor in the validation of the genome-scale metabolic model and provide support in the dry lab experiments.</p>
<hr>
@@ -51,6 +50,7 @@
<p>Prof. Marcelo Brocchi´s team: granted us permission to use his electroporator.</p>
<p>Prof. André Damasio, Fernanda Lopes de Figueiredo and Laboratory of Enzymology and Molecular Biology (LEBIMO): helpful insights, discussions and helped with carbohydrate quantification.</p>
<p>Laboratory of Genomics and Bioenergy (LGE): supplied equipment for electroporation and glucose measurement.</p>
+ <p><b>Fellipe da Silveira Bezerra de Mello</b> acted as team consultant in key stages of Cellulopolis. Provided lab and project support in the wet and dry lab experiments.</p>
<hr>
diff --git a/wiki/pages/awards.html b/wiki/pages/awards.html
index b1204104fd2bd7de39257f0e61ccfe02253195cc..732d189fd9cb639d223b975537defed569cd98db 100644
--- a/wiki/pages/awards.html
+++ b/wiki/pages/awards.html
@@ -7,12 +7,10 @@
<div class="row mt-4">
<div class="col">
- <div class="bd-callout bd-callout-info">
- <h4>Awards</h4>
- <p>Text.</p>
- <hr>
- <p>Text.</p>
- </div>
+ <h1> COMING SOON </h1>
+
+ <p> After the competition the team plans to extend the project and continue with its real-world application. we are already in contact with organizations working with startup incubating ideas. Our plans involve turning the project into a real product that can be patented and sold. This was we will reassure that we’re constantly reaching for our goal. That is to make something useful that’s also good for the planet.</p>
+
</div>
</div>
diff --git a/wiki/pages/communication.html b/wiki/pages/communication.html
index 599b67128352dfee861c902799ec33ae92979db2..add7602f5c0f27169294fb4baffe4e16c2808ad9 100644
--- a/wiki/pages/communication.html
+++ b/wiki/pages/communication.html
@@ -1,7 +1,7 @@
{% extends "layout.html" %}
{% block title %}Communication{% endblock %}
-{% block lead %}Develop and implement education, science communication, and/or outreach materials related to synthetic biology.{% endblock %}
+{% block lead %}{% endblock %}
{% block page_content %}
@@ -16,6 +16,9 @@
<p>The possibility of participating in iGEM brought a number of learnings, among them the need to share knowledge with peers to achieve high impact results, polished from different perspectives. With this in mind and the desire to preserve a tradition of continuity in synthetic biology activities within our academic community, we founded the Synthetic Biology Club: a multidisciplinary entity determined to cultivate a nucleus of synthetic biology studies within our campus seen from different lenses, ranging from biology to the social and philosophical spheres. In this environment, besides discussing possible social interventions such as the ones we did in schools, we also discuss ethical issues related to the practice of synthetic biology. To us, social engagement and the articulation of science with the relevant issues of our time are indispensable for the popularization and prosperity of science. And that is one of our priorities. </p>
<p>Far beyond a single iGEM project, our expectations are that the Club will incubate new teams for future editions of the competition and remain active in the scientific and ethical debate concerning synthetic biology and its related areas.</p>
<h1>Seeds Project - School Madre Cabrini</h1>
+<figure class="figure-left">
+ <img class="pages-img" src="https://static.igem.wiki/teams/4435/wiki/pages/education/visita-madri.png">
+</figure>
<p>Our first experience of dissemination and more direct contact with elementary school students took place at Colégio Madre Cabrini, in São Paulo. In which we tried to incubate the investigative spirit in the students, working not only in iGEM, but in the scientific practice itself. Additionally presenting to the students, what the competition is about and the project we were developing at the time about organic waste disposal through a language accessible to them. We coordinated a critical and scientific thinking activity in the high school classrooms: the students had the time of one class - approximately 50 minutes - to think about the possibilities of synthetic biology and the initial idea of a project that would address an everyday problem of their perception.</p>
<p>As we went deeper into iGEM, the goal was to incubate seeds in the minds of the students, of the capabilities of science in solving modern problems, in the hope that these seeds will germinate to form new scientists: agents with transformative potential necessary for social development.</p>
<h1>UNICAMP's Open Doors Weekend (UPA)</h1>
diff --git a/wiki/pages/entrepreneurship.html b/wiki/pages/entrepreneurship.html
index 207ef85e14f63a0c5e5e858cd114506833ab14b9..06b8de651252e29a0dc8ffbfe8f8f63ac0f87c1e 100644
--- a/wiki/pages/entrepreneurship.html
+++ b/wiki/pages/entrepreneurship.html
@@ -14,7 +14,7 @@
<p>The financial and technological positive impact of the COVID-19 pandemic on the Biotech sector carried out by the investigations of research centers, companies, and startups, in search of a solution for the state of emergency, placed the Biotech market as one of the most emerging worldwide. Here we carry out a detailed analysis of our three main markets: vegetable cellulose, synthetic biology, and bacterial cellulose using The Brazilian Tree Industry (IBA) and the stock market data (NASDAQ) as a reference to analyze the feasibility of the implementation of our project as a company within the economic market. To diagnose the critical points of our company we used SWOT-type business tools. Furthermore to assess the sales process and decision-making between lead/customer we build a canvas-type business model and we use funnel and pipeline tools to define the contact iteration channels with the Customer Relationship Management (CRM). With all this analysis we made the organization chart of our company CELLULOPOLIS as an innovative and sustainable product for market implementation.</p>
- <h1>Profile of the cellulose Market in Brazil</h1>
+ <h2>Profile of the cellulose Market in Brazil</h2>
<p>The cellulose and paper sector plays an essential role in the national and international economy. This is due to the revenue generated, the high investments, the impact that this sector has on the other various economic sectors, before and after its production chain, as well as its influence on the generation and consumption of energy.</p>
@@ -27,11 +27,11 @@
<figcaption>Cellulose production, importation, and exportation</figcaption>
</figure>
- <h1>Cellulose and Paper Market</h1>
+ <h3>Cellulose and Paper Market</h3>
<p>Although there is the idea of replacing paper with other technologies, the sector has shown growth over the years. The demand for paper has shown an increase that is proportional to the development of the population. In addition, there is a worldwide trend towards replacing plastic with paper, due to the shorter decomposition time of paper. In line with this trend, the R&D areas of cellulose and paper companies have sought solutions in the development of new materials, the use of co-products, and new markets for the application of cellulose. There is also a constant investment in improving processes, aiming at gains in production and quality in the final product, to serve an increasingly demanding customer, who prioritizes the consumption of certified and environmentally responsible products.</p>
- <h1>Profile of the global synthetic biology market</h1>
+ <h2>Profile of the global synthetic biology market</h2>
<p>The international synthetic biology market is in a strong long-term uptrend, giving good scope for future development.The number of startups that have synthetic biology listed as a primary activity and with capital above USD 100,000 doubled between 2017 and 2020, which indicates not only broad economic success in the multi-company market but also a large growth of professionals in the area. In addition, 8 of the 10 companies with the highest market value in the industry were founded in the last decade.</p>
@@ -46,7 +46,7 @@
<p>The Synthetic Biology market is on the rise until 2021 according to the movement in NASDAQ ALL-TIME SYNBIO STOCKS points.</p>
- <h1>Global acting of synbio-related companies in 2020/2021 </h1>
+ <h2>Global acting of synbio-related companies in 2020/2021 </h2>
<figure class="pages-img">
<img src="https://static.igem.wiki/teams/4435/wiki/entrepreneurship/fig3.png">
@@ -60,7 +60,7 @@
<p>The first peak and crash of investments in the Synthetic Biology sector within the NASDAQ Stock Market occurred between 2015 and 2016 with the widespread notification and development of alternatives to fossil fuels through the production and synthesis of biodiesel. Currently, the subject has returned to the agenda through the synthesis and manipulation of marine organisms, which could lead to a new peak in investments as international oil prices soar. There is also heavy investment in the food sector despite the breadth of technology allowing a wide variety of services and products.</p>
- <h1>Bacterial cellulose market profile</h1>
+ <h2>Bacterial cellulose market profile</h2>
<p>The global bacterial cellulose production market has shown a strong rise since 2018 and the perspective is to increase even more until 2027. The means of production specifically in Brazil have a constant investment in improving processes, aiming at gains in production and quality in the final product, to serve an increasingly demanding customer, who prioritizes the consumption of certified and environmentally responsible products.</p>
@@ -71,14 +71,14 @@
<figcaption>Microbial and Bacterial Market Size prospeccion.</figcaption>
</figure>
- <h1> SWOT analysis</h1>
+ <h2> SWOT analysis</h2>
<figure class="pages-img">
<img src="https://static.igem.wiki/teams/4435/wiki/entrepreneurship/imagen5.png">
<figcaption>Diagram of the company’s SWOT analysis considering the market presence</figcaption>
</figure>
- <h1>Market analysis conclusion and future perspectives</h1>
+ <h2>Market analysis conclusion and future perspectives</h2>
<p>After evaluating the three potential markets we consider our project as one of the most promising in the educational, health, and industry sectors, positively impacting the national and international economy. Then, after identifying our strengths and weaknesses with the Swot tool analysis, we decided to go one step further with the creation of the organization of our company. Based on all this we plan to be part of the incubation program for young companies of the INOVA Unicamp group, where we can interact with universities, researchers, and companies in search of future partners.</p>
<h1>Product</h1>
diff --git a/wiki/pages/hardware.html b/wiki/pages/hardware.html
index 9bd63e2bfa112d887c62b7d458f5c2cccf8c36d5..b4696ca34f10cea8a7807a5719f026534bcf4fbe 100644
--- a/wiki/pages/hardware.html
+++ b/wiki/pages/hardware.html
@@ -5,11 +5,54 @@
{% block page_content %}
-<div class="row mt-4">
- <div class="col">
- <embed src="https://static.igem.wiki/teams/4435/wiki/pages/hardware/hardware-pdf.pdf" width="500" height="375"
- type="application/pdf">
- </div>
-</div>
+<h1>Bacterial cellulose as a scaffold for tissue engineering</h1>
+<h2> How our biopolimer can be used to form tissues!</h2>
+
+
+
+<p>Bacterial cellulose (BC) is a versatile material that could potentially be used as a scaffold for tissue engineering. Therefore, our team is optimizing Komagataeibacter for the production of BC and testing the development of molds that would allow the growth of BC sheets in the correct format for tissue culture and subsequent 3D organ assembly. To that end, we converted the surface of a complex object into a 2D shape, printed the perimeter as a “cookie cutterâ€, and used it as a template for bacterial cellulose sheet production.</p>
+
+<p>Unfolding the shape of a sphere into a 2D object</p>
+
+<p>The surface of 3D objects can be readily unfolded into 2D objects provided that the object is subdivided into segments with single curvatures. For example, the surface of a cylinder can be unfolded into a rectangle. Objects with curvature in multiple directions, such as spheres, pose a greater challenge. However, there is a great number of published works that suggest 2D shapes that reasonably capture the surface of a sphere. </p>
+<p>Demaine and colleagues (Computational Geometry 42 (2009) 748-757) defined a number of 2D shapes to represent the area of a 3D sphere, minimizing area (compatible with small printing beds) or minimizing the perimeter (fewer cuts and joints). Due to the constraints of a small 3D printer the Unicamp_Brazil team had available, we selected a 2D k-petal model with 6 petals for this project (Figure 1, extracted from Demaine et al, 2009).</p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/hardware/hardware-shape.png">
+ <figcaption>Figure 1. The perimeter of the surface of a sphere (blue) is represented by 6 petals, occupying the smallest possible area whilst allowing a good wrapping of the sphere.</figcaption>
+</figure>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/hardware/3dmodeltransparent.gif">
+ <figcaption>Figure 2. </figcaption>
+</figure>
+
+<p>Delimiting the growth of BC for assays in 24-well plates</p>
+
+<p>Initial experiments for the growth of animal cells using cellulose as a scaffold require cutting the sheets in appropriate proportions to fit them into plates of cell culture medium. The growth of cellulosic fibers in different formats, for later cutting of what would be used, causes destandardization in the final product, in other words, we obtain a portion of cellulose with variable thickness and irregular surface that makes cell cultivation difficult. To minimize the effects of these variables on the culture, we built a mold that allows the production of cellulose in standardized formats ideal for fitting into 24-well culture plates, avoiding variations in the cellulosic surface.</p>
+
+
+<h2> Bioreactors</h2>
+
+<p>The project design aims to engineer strains with a bimodal growth pattern, divided in biomass accumulation stage and Bc production stage. To achieve this we are engineering a K. rhaeticus AF1 where cellulose synthase genes are off under dark and expressed by the use of blue light. Therefore, we devised a bioreactor to grow Komagataeibacter in an environment where there is no light and switch to an inducible environment when needed.</p>
+
+<h2>Growth optimization.</h2>
+
+<p>For growth optimization, some criteria were considered in the design of the bioreactor. As was seen in the math models for growth the Komagataeibacter is obligately aerobic, so the oxygen supply is very important. Another point discussed was the cost of construction. Fitting the bioreactor with an impeller would significantly increase the cost, therefore, to allow for mixing and oxygen supply, it was decided to build an air lift bioreactor.</p>
+
+<h2>Production optimization.</h2>
+
+<p>We investigated different induction alternatives that would allow the expression of cellulose synthate (bcs) when we desired, however, the costs of inducers for the characterized Komagataeibacter promotors are prohibitive. Hence, we explored the scientific literature and encountered a recent paper describing a single-component light-sensitive transcriptional repressor. By using a light-sensitive promoter (LexRO) the production of BC can become cheaper. However, to induce the reaction efficiently, a bright place for the reaction is needed. </p>
+
+<p>To unite the two needs the team developed two pieces of hardware. The first part of the growth will be done in a low-cost reactor. The first part of the growth will be done in a low-cost reactor, made out of common laboratory materials. For the fermentation vessel, a 1-liter reagent bottle was chosen. All inlets were located in the lid, following the design of commercial bioreactors. For the air inlet, a small aquarium compressor was used (4 L/min). For prototyping purposes, this reactor was tested with E. coli, due to its fast growth rate. </p>
+
+<p>The first tests were successful, opening opportunities for improvement in some parts. Our goal is to couple both pieces of hardware: after leaving the bioreactor (biomass accumulation), the biomass will be directed to the second stage, the photobioreactor, which will activate cellulose production in defined molds.</p>
+
+<p>With this integration of the bioreactor with the photobioreactor using peristaltic pumps. Thus making cellulose production a continuous process increasing the productivity.</p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/hardware/hardware-bioreator.png">
+ <figcaption>Figure 3. </figcaption>
+</figure>
{% endblock %}
diff --git a/wiki/pages/human-practices.html b/wiki/pages/human-practices.html
index 5c9a4f74984011c01b676c0cdf13a39948e454f1..474ce41adaa5045f10e68590b880d949dddd4277 100644
--- a/wiki/pages/human-practices.html
+++ b/wiki/pages/human-practices.html
@@ -21,6 +21,8 @@
<p>The company <b>Corpus Saneamento e Obras</b> gave us the first opportunity to visit their recycling centers and learn how the workflow for reworking the waste works. The visit to the waste management laboratory of Professor Miriam Gonçalves Miguel, a Unicamp researcher in solid waste management, was also important in this sense. Their availability was fundamental to building the first understanding of what we were dealing with and what are the factors that add complexity to the problem of inappropriate management of these materials.</p>
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/human-practices/expo.jpg" class="figure-left">
+
<p>Another important contact with the theme was the participation in the <b>"Waste Expo 2021"</b> event, a fair held annually in São Paulo that brings together a variety of companies from the sanitation and solid urban waste management sector. Just as companies take the opportunity to probe new technologies and opportunities, we also took the chance to network with companies in the sector and understand the market dimension of our project.</p>
<p>This first couple of feedback gave us valuable insights on the way forward. We found out that organic "contaminants", especially grease, hinder the recycling process, and a pre-treatment of solid waste is still costly and little implemented in recycling centers. We also found that for some types of waste the biggest obstacle is not recycling itself, but the logistical difficulty of transporting large volumes of the material in front of its low density, as is the case of expanded polystyrene (EPS). Besides being a material difficult to reuse, large amounts of polystyrene require a costly logistics operation for companies in the sector since 95% of the volume of this plastic is air, which makes it more expensive and unprofitable to recycle.</p>
diff --git a/wiki/pages/implementation.html b/wiki/pages/implementation.html
index ac8f900ae540c766ddaffc29b4f0e7440cae7300..bb161d2f5865241c4e87b104daeb083f953ddd4d 100644
--- a/wiki/pages/implementation.html
+++ b/wiki/pages/implementation.html
@@ -37,14 +37,15 @@
<h2>Small scale implementation challenges</h2>
<img src="https://static.igem.wiki/teams/4435/wiki/proposed-implem/imagen2-png.png" class="figure-left">
<p>As challenges, we can mention the great handling difficulty that the Komagataeibacter bacterium provides. This organism takes 3 days to grow, which delays the process a little depending on demand and urgency. Komagataeibacter is a reasonably expensive bacterium to maintain, because its culture medium, HS (Hestrin; Schramm, 1954), commonly used in laboratories, carries a large amount of glucose, being 50g, while most common media takes around 2g - 4g. It is also valid to bring the complication in automating the removal of the cellulose blanket, after its production, in addition to the wide obstacle encountered in making this whole process, of cellulose production, become continuous, for reasons of possible contamination and also the impasse encountered at the time of removal of the cellulose produced, considering that when this polymer is removed, the cell is also removed.</p>
+ <img src="https://static.igem.wiki/teams/4435/wiki/proposed-implem/imagen3-celulosa.png" class="figure-left">
</div>
</div>
<div class="row mt-4">
<div class="col">
<h2>Manufacturing scale implementation challenges</h2>
- <img src="https://static.igem.wiki/teams/4435/wiki/proposed-implem/imagen3-celulosa.png" class="figure-left">
-
+
+ <img src="https://static.igem.wiki/teams/4435/wiki/proposed-implem/imagen4-custumerpng.png" class="figure-left">
<p>We focused primarily on reducing times within the production system as well as response times from suppliers and customers, thus our process of choice was utilizing the 5 principles of manufacturing (commonly referred to as <b>lean manufacturing</b>). This process was very useful to make us understand how we would conduct our manufacturing processes and to always keep aiming for perfection. most of these steps were supported further by data collected in our process and market research. <b>The challenges</b> we will face in each of these steps will be mapped and organized in a way that mistakes can be fixed as quickly and as efficiently as possible. </p>
</div>
@@ -59,7 +60,6 @@
<p>According to the prospect made in the market analysis and in the strategic planning of our business (see entrepreneurship), our clients are corporations involved in the pharmaceutical and cosmetics industry mainly. However, our customers are different from our end consumers for a number of reasons. Since our customers are companies that would use our product in their services, our true end consumers are the people who use these companies' services. For example, if our bacterial cellulose is sold to a hospital that uses it to treat burns of a patient, our customer (who bought our product) would be the hospital, but who would really be consuming this would be the patient (hospital customer).</p>
- <img src="https://static.igem.wiki/teams/4435/wiki/proposed-implem/imagen4-custumerpng.png" class="figure-left">
<p>In this way, we define our end consumers as the clients of our clients. People involved in accidents with burns in low-income hospitals, who would make the best use of our products sold at low cost, or consumers of cosmetics containing our cellulose (purchased by one of our customers). With our cellulose production we can deliver the final product to hospitals, companies, and laboratories in the form of gel, ointment, scaffold, blocks or other conditions required by the customer.</p>
</div>
</div>
diff --git a/wiki/pages/notebook.html b/wiki/pages/notebook.html
index 2c86b3c667505c69cda65abda9d1cb852b98941e..7a56ac5f88ad20b9f586c0ea2888e1d80b8d2978 100644
--- a/wiki/pages/notebook.html
+++ b/wiki/pages/notebook.html
@@ -1,34 +1,536 @@
{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
-{% block lead %}{% endblock %}
+{% block lead %}The step by step of our lab processes{% endblock %}
{% block page_content %}
-<div class="row mt-4">
- <div class="col-lg-8">
- <h2>What should this page contain?</h2>
- <hr>
- <ul>
- <li>Chronological notes of what your team is doing.</li>
- <li>Brief descriptions of daily important events.</li>
- <li>Pictures of your progress.</li>
- <li>Mention who participated in what task.</li>
- </ul>
- </div>
- <div class="col-lg-4">
- <h2>Inspirations</h2>
- <hr>
- <ul>
- <li><a href="http://2018.igem.org/Team:Munich/Notebook">2018 Munich</a></li>
- <li><a href="https://2019.igem.org/Team:Georgia_State/Notebook">2019 Georgia State</a></li>
- <li><a href="https://2019.igem.org/Team:Newcastle/Notebook">2019 Newcastle</a></li>
- <li><a href="https://2020.igem.org/Team:IISER-Pune-India/Notebook">2020 IISER Pune India</a></li>
- <li><a href="https://2020.igem.org/Team:Lund/Notebook">2020 Lund</a></li>
- <li><a href="https://2020.igem.org/Team:NOVA_LxPortugal/Notebook">2020 NOVA LxPortugal</a></li>
- <li><a href="https://2020.igem.org/Team:RDFZ-China/NoteBook">2020 RDFZ China</a></li>
- </ul>
- </div>
-</div>
+<h1>March to June 2021</h1>
+
+<p>Eduardo Menoti Silva, Gustavo Seguchi, Julia Ferreira Oliveira, Larissa Monteiro, Luiza Hesketh Gomes, and Michele De Vuono Geismar Petineli decided to participate in iGEM 2022</p>
+
+
+<h1>July to December 2021</h1>
+
+
+<p>Recruit members outside the Institute of Biology</p>
+<p>Vistis to waste management facilities, meeting, meetings and more meetings</p>
+<p>First Styropolis project draft: Engineer bacteria to convert polystyrene into PHA ot PHB</p>
+<p>Discussions with past iGEM teams</p>
+<p>Design visual identity for the team</p>
+<p>Design documentation for sponsors</p>
+<p>Contact sponsors</p>
+
+
+
+<h1>January to March 2022</h1>
+
+
+<p>Preliminary trials with different solvents to shrink polystyrene</p>
+<p>Acquire <i>Streptomyces</i>, <i>Bacillus</i> and <i>Lactobacillus</i> as candidate chassis</p>
+<p>Request Freegenes toolkit</p>
+
+<img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/limoneno.gif">
+
+<p>Cultivation of mealworms fed on polystyrene as a source of gut bacteria expressing polystyrene degrading enzymes</p>
+
+<img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/human-practices/larvas.png">
+
+<h1>April 2022</h1>
+
+<p>Pay iGEM</p>
+<p>Order plastic ware, enzymes, kits and chemicals</p>
+
+
+<h1>May 2022</h1>
+
+<p>New interviews and calculations revealed that Styropolis would not be beneficial to society. </p>
+
+<p>PANIC!!!! Back to the drawing board. </p>
+
+<p> Half of the team left</p>
+
+<p>Why not work with a different bacterium that can convert other waste product into a high value product?</p>
+
+<p>Open selection process for Unicamp_brazil team and Synthetic Biology club</p>
+
+
+<h1>June 2022</h1>
+
+
+<p>Recruit new team members (Unicamp_Brasil and Synbio club)</p>
+
+<p>Abundant waste product in our region: orange pulp</p>
+
+<p> Definition of the Chassis:<i> Komagataiebacter</i> </p>
+
+<p>Product: bacterial cellulose </p>
+
+<p> Challenge: how can we engineer bacteria to produce more cellulose at lower cost?</p>
+<p> iGEM distribution kit stuck at customs </p>
+<p>Consumables arriving at the lab </p>
+
+
+<h1>July 2022 : first week</h1>
+
+<p>iGEM distribution still stuck at customs (daily calls to UPS, customs, ministry of science, and ministry of agriculture to try and free the samples). </p>
+<p>design, create Gcode and print the first mold for BC production in defined shapes. </p>
+
+<p>Consumables arriving at the lab</p>
+
+<h1>July 2022: second week </h1>
+
+<p>Michele and Larissa travel to Araraquara for hands-on training in <i>Komagataeibacter</i> cultivation and BC production</p>
+
+<p><i>Komagataeibacter rhaeticus</i>AF1 and <i>K. Medellinensis</i> ID13488 arrive at Unicamp. </p>
+
+<p>iGEM distribution still stuck at customs (daily calls to UPS, customs, ministry of science and ministry of agriculture to try and free the samples)</p>
+
+<p>Design engineering strategy (select relevant plasmids from toolkit and begin custom primer and gene design)</p>
+
+<p>Preparation of the media</p>
+
+<p>Test different protocols for preparation of ultracompetent E. coli DH5alpha cells (best competence achieved with cells prepared with Inoue buffer (stored at -80oC) (protocol by Sambrook and Russel). </p>
+
+<p>DH5aplha transformation:</p>
+<p>Slowly thaw competent cells on ice</p>
+<p>Add 2-5 uL of DNA to 40-60 uL of competent cells in a fresh sterile tube</p>
+<p>Incubate for 30 min</p>
+<p>Heat shock at 42oC for 45 sec</p>
+<p>Place on ice</p>
+<p>Add 500 uL of SOC</p>
+<p>Recover by incubation at 37oC for 1h</p>
+<p>Spin for 2 min at 10000 rpm </p>
+<p>Discard supernatant by inversion</p>
+<p>Ressuspend cell pellet in the residual liquid and plate onto selective LB media</p>
+
+<p>Calculate transformation efficiency: 2,8 x 106 colonies/ug DNA</p>
+
+<h1>July 2022: third week</h1>
+
+<p>Distribution kit arrived!!!</p>
+<p>Prepare LB (10 g tryptone; 10 g NaCl; 5 g yeast extract; add dH2O; adjust pH to 7; 15 g agar; complete to 1 L) and HS (50 g glucose; 0,75 g MgSO4; 2 g KH2PO4; 4g yeast extract; 20 ml ethanol; complete to 1 L with dH2O; sterile filter) </p>
+<p>Transform DH5alpha with plasmids from distribution plate 1 positions:</p>
+<p>1c, 1e, 1g, 1i, 1k, 1m (chloramphenicol)</p>
+<p>2k, 2m (kanamycin)</p>
+<p>3l, 3n, 3p (chloramphenicol)</p>
+<p>4a (kanamycin)</p>
+<p>5b, 5d, 5f, 5h, 5j, 5l, 5n, 5p (chloramphenicol)</p>
+<p>10i, 10k, 10m, 10o (kanamycin)</p>
+<p>12e, 12g, 12i, 12k, 12m, 12o, 14a, 14c, 14e (chloramphenicol)</p>
+<p>Ressuspend DNA from iGEM plates in 10 uL ddH2O and use 2 uL to transform 40 uL of competent DH5alpha</p>
+
+<p>Inoculate K. rhaeticus AF1 and K. medellinensis in HS with cellulase for transformation - cellulase not working - repeat</p>
+
+<h1>July 2022: fourth week</h1>
+
+<p>Make glycerol stocks of iGEM plasmids in DH5aplha (750 uL culture + 250 uL of 60% glycerol)</p>
+<p>Miniprep (Bioflux kit) all relevant iGEM plasmids</p>
+<p>Inoculate transformantes for Interlabs experiments 1, 2 and 3 (50 mL tubes; 2 independent cultures each)</p>
+<p>Perform all 3 Interlabs experiments according to protocols provided</p>
+
+<p>Finalize strain, primers and gene designs</p>
+<p>Inoculate K. rhaeticus AF1 and K. medellinensis in HS with new cellulase for transformation; measure ODs:
+1. 0,548
+2. 0,673
+3. 0,687
+4. 0,523
+5. 0,438</p>
+<p>Adjust OD to 0,04 and incubate for 24 h; measure ODs:
+1. 0,334
+2. 0,335
+3. 0,337
+4. 0,255
+5. 0,320</p>
+<p>Measure OD and incubate for another 24 h;
+1. 0,831
+2. 0,492
+3. 0,493
+4. 0,5
+5. 0,490</p>
+<p>Repeat due to contamination and after 3 days, prepare electrocompetent according to protocol developed by iGEM Imperial team 2014. Transform plasmids BBa_J428346, BBa_J428347 and BBa_J428349. </p>
+
+<h1>August 2022: first week </h1>
+
+<p>Evaluate <i>Komagataeibacter</i> transformations. No difference between bacteria transformed with plasmids or negative control. Conclusion: contamination. Repeat.</p>
+<p>Place gene and primer synthesis order with IDT</p>
+
+<p>Grow membranes in shapes</p>
+
+<p>Repeat Interlabs experiments</p>
+
+
+<h1>August 2022: second week </h1>
+
+<p>gene and primer synthesis order with IDT finally accepted</p>
+
+<h1>August 2022: fourth week </h1>
+
+<p>Transform DH5alpha with part BBa_J04450 for shipment to iGEM UFMG team (LB chloramphenicol)</p>
+<p>Transform DH5alpha with FLP (iGEM distribution kit plate 2 position E6) (LB Kan)</p>
+<p><i>K. rhaeticus</i> AF1 transformation……FAIL</p>
+
+
+<h1> September 2022: second week </h1>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura1.png">
+ </figure>
+
+<p>As IDT parts are not arriving on time, design new strategy and transform DH5alpha with new plasmids from the iGEM distribution plates:</p>
+<p>Plate 1 positions:
+15i, 15c, 15g, 13o, 15a, 15e, 23g, 9p, 13m, 23a, 13a, 16g, 13g, 13c, 13e (CmR)
+2k, 2m, 4a (KanR)
+6k, 6m, 8a (SpecR)
+13p, 1o (CmR)
+10k, 10m, 10o, 12a, 12c, 10g (CmR)</p>
+<p>Plate 2 positions:
+D4, F6, D8 (AmpR)
+11i (CmR)
+11k (KanR)
+11m (tetracycline)
+6E (AmpR)</p>
+<p>Design and order primers for synthesis with Exxtend. Primers for marker swap of JUMP plasmids (with broad spectrum ori). </p>
+<p>PCR products of AmpR (2x) and CmR (2x) with primers with adaptors for ligation with Jump backbones (Purify PCR products with BioFlux kit)</p>
+
+
+<p>Transform K. rhaeticus AF1……FAIL</p>
+<p>Grow K. rhaeticus AF1 and K. medellinensis in different media to quantify BC production</p>
+<h1>September 2022: third week </h1>
+
+<p>Minipreps of toolkit parts for new construct design (low yield; shaker kept at 30oC due to Komagataibacter cultures; probably low yield due to poor E.coli growth; solution: incubate for longer and with larger volume in larger tubes)</p>
+
+<p>Weight BC wet sheets:
+AF1- wet sheet 1,113 g
+AF1 – wet sheet 1,1949 g
+Medel – wet sheet 1,1754 g</p>
+<p>Dry sheets:
+AF1- 0,0163 g
+AF1 – lost
+Medel – 0,0170 g </p>
+
+<p>Repeat PCR of backbones for marker swap: fail. In the same gel the last 4 lanes show the PCR products of AmpR (2x) and CmR (2x) with primers with adaptors for ligation with Jump backbones. </p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura2.png">
+ </figure>
+
+<p>New PCR of Jump plasmids. Mix per reaction (prepare on ice; prepare x8 and distribute into 0,2 ml strips for gradient)
+5 ul of 5x Q5 reaction buffer
+2 ul of 2,5 mM dNTPs
+0,125 ul of 100 uM primer F
+0,125 ul of 100 uM primer R
+0,1 ul template plasmid (10 ng/ul)
+0,25 ul Q5 DNA polymerase
+5 ul Q5 high GC enhancer
+12,5 ul ddH2O</p>
+<p>Reaction conditions:
+98oC for 1 min
+98oC for 15 sec
+50-60oC for 30 sec
+72oC for 1,5 min
+back to step to 30x
+72oC for 5 min
+hold at 4oC</p>
+<p>All reactions worked!!! (Purify PCR products with BioFlux kit). Expected band sizes:</p>
+<p>2k (BBa_J428346)– 2,7 kb</p>
+<p>2m – 2 kb</p>
+<p>6k (BBa_J428366)– 2,7 kb</p>
+<p>6m – 2 kb
+2 lanes of failed site directed mutagenesis</p>
+<p>4a – 4,2 kb</p>
+<p>8a – 4,2 kb</p>
+<p>Quantification after purification:
+6k – 29,15 ng/ul
+6m – 35 ng/ul
+8a – 36,15 ng/ul</p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura3.png">
+ </figure>
+
+<p>Attempt Goldengate – fail</p>
+
+<p>Transform K. rhaeticus AF1……FAIL</p>
+
+<p>1% agarose gel of miniprep of new parts</p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura4.png">
+ </figure>
+
+<p>Agarose gel of PCRs of 5´homology arms (K. rhaeticus AF1)</p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura5.png">
+ </figure>
+
+<h1>September 2022: fourth week </h1>
+
+<p>GoldenGate to combine JUMP plasmids 2k, 2m, 4a, 6k, 6m, 8a with AmpR or CmR</p>
+<p>0, 5 ul T4 DNA ligase
+2 ul 10 x T4 ligase buffer
+0,5 ul SapI
+3 ul plasmid backbone
+1 ul AmpR or CmR</p>
+<p>Incubate at 37oC for 1h, 16oC 5 min, 55oC 5min, hold at 4oC</p>
+<p>Transform 5 ul of product into DH5alpha. Select on LB amp or LB chloramphenicol (it worked!!!)</p>
+<p>Inoculate for minipreps</p>
+<p>Minipreps on 26/09</p>
+
+<p>Transform DH5alpha with iGEM plasmids 2c, 2e and 2g (distribution plate 1) – plate onto LB kan</p>
+
+<p>26/09. Synthetic LexRO and FLPe arrived!!!!!!</p>
+
+<p>Golden Gate LexRO and FLPe into plasmid 13p (iGEM backbone) – fail</p>
+
+<p>(26/09)</p>
+<p>Measure OD of Komagataeibacter cultures to adjust OD:
+<p><i>K. medellinensis</i>: 0,354
+<i>K. rhaeticus</i> AF1: 0,605</p>
+
+<p>BRICS measurement: </p>
+<p>5,2</p>
+<p>5,2</p>
+<p>3,8
+3,7
+3,7
+3,5</p>
+
+<p>(27/09)</p>
+<p>OD for preparation of competent <i>K. rhaeticus</i>:</p>
+<p>1. 0,282 0,346 0,412</p>
+2.<p> 0,260 0,382 0,625</p>
+3. <p>0,283 0,384 0,637</p>
+4. <p>0,263 0,542 0,709</p>
+5. <p>0,263 0,364 0,797</p>
+
+<p>BRICS measurement: </p>
+<p>5,1</p>
+<p>5,2</p>
+<p>3,7</p>
+<p>3,2</p>
+<p>3,3</p>
+<p>3,2</p>
+
+<p>(28/09)</p>
+<p>BRICS measurement: </p>
+<p>5,2</p>
+<p>5,1</p>
+<p>3,2</p>
+<p>3,0</p>
+<p>3,0</p>
+<p>3,0</p>
+
+<p>(29/09)</p>
+<p>BRICS measurement: </p>
+<p>5,1</p>
+<p>5,1</p>
+<p>3,2</p>
+<p>3,2</p>
+<p>3,1</p>
+<p>3,1</p>
+
+<p>Transform K. rhaeticus AF1 and K. medellinensis……maybe….. no…. it's a FAIL</p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura6.png">
+ </figure>
+
+<p>(28/09) IDT primers and short parts arrived!!!!!! Ressuspend plasmid DNA with 40 uL of H2O for 100 ng/ul stocks</p>
+
+<p>Golden Gate of new parts into iGEM backbone</p>
+<p>TU4p_RBS</p>
+<p>TU_term</p>
+<p>TU3p_c</p>
+<p>TU3p_a</p>
+<p>Dgc1</p>
+<p>TU3p_b</p>
+<p>TU_RBS</p>
+<p>(Did not work)</p>
+
+<p>PCR of Komagataeibacter genes with new IDT primers</p>
+<p>galU</p>
+<p>pgm (1,7 kb)</p>
+<p>ndp (0,5 kb)</p>
+<p>GK</p>
+<p>TU1a (homology arm) (1,4 kb)</p>
+<p>TU1b (homology arm) (1,4 kb)</p>
+<p>TU4a cds (homology arm)</p>
+<p>TU4b cds (homology arm) (1,4 kb)</p>
+
+<p>PCR mix:</p>
+<p>0,25 ul primer F</p>
+<p>0,25 ul primer R</p>
+<p>4 ul dNTP 2,5 mM</p>
+<p>10 uL Q5 buffer</p>
+<p>0,1 ul genomic DNA</p>
+<p>0,5 ul Q5 DNA polymerase</p>
+<p>10 uL PCR enhancer</p>
+<p>complete to 50 uL with H2O</p>
+<p>PCR conditions:</p>
+<p>98oC for 5</p>
+<p>98oC for 30 sec</p>
+<p>55oC for 20 sec</p>
+<p>72oC for 45 sec</p>
+<p>back to 2 30x</p>
+<p>72oC 5min</p>
+<p>hold at 4oC</p>
+
+<p>Gel of PCR from genomic DNA of K. rhaeticus AF1:</p>
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura7.png">
+ </figure>
+
+<p>Gel of PCR from genomic DNA of K. medellinensis: </p>
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura8.png">
+ </figure>
+
+<p>Purify successful PCRs and optimize other with gradient</p>
+<h1>October 2022: first week </h1>
+<p>(01/10)</p>
+<p>BRICS measurement: </p>
+<p>5,1</p>
+<p>5,05</p>
+<p>3,2</p>
+<p>3,1</p>
+<p>3,05</p>
+<p>3</p>
+
+<p>(04/10)</p>
+<p>BRICS measurement: </p>
+<p>5,1</p>
+<p>5,1</p>
+<p>3,2</p>
+<p>3,1</p>
+<p>3,1</p>
+<p>3,1</p>
+
+<p>Gradient PCR (50-60oC annealing temperature) of genomic DNA AF1 for amplification of galU, GK and homology arms TU1b and TU4a; and genomic DNA medellinensis for amplification of galU, GK and homology arms TU1a , TU1b and TU4a.</p>
+
+<p>Gel of PCR from genomic DNA of K. medellinensis: </p>
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura9.png">
+ </figure>
+
+
+ <figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura10.png">
+ </figure>
+
+
+ <figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura11.png">
+ </figure>
+
+
+
+<p>Conclusion: optimum annealing temperature for galU F and R is from 60-65oC; GK F andR is from 60-65oC; Homology arm primers- ideal annealing temperature between 67.9 and 70.1oC. </p>
+
+<p>Transform DH5alpha with iGEM plate 1 plasmids 18m and 18o (type CDS); 16c and 16g (type promotor); 16e and 16o (type RBS); 18c and 20c (type terminator).</p>
+
+<p>Construct parts parts BBa_K4435315, BBa_K4435316, BBa_K4435317:</p>
+<p>PCR mix:</p>
+<p>0,25 ul primer AdapterTUred_F</p>
+<p>0,25 ul primer AdapterTUred_R</p>
+<p>4 ul dNTP 2,5 mM</p>
+<p>10 uL Q5 buffer</p>
+<p>0,1 ul mniprep 13p</p>
+<p>0,5 ul Q5 DNA polymerase</p>
+<p>complete to 50 uL with H2O</p>
+<p>PCR conditions:</p>
+<p>98oC for 5´</p>
+<p>98oC for 20 sec</p>
+<p>54 to 66oC for 20 sec</p>
+<p>72oC for 45 sec</p>
+<p>back to 2 30x</p>
+<p>72oC 5min</p>
+<p>hold at 4oC</p>
+<p>PCR worked well! Purify with Bioflux (elute in 30 uL) and proceed to Goldengate</p>
+
+<p>GoldenGate of red adapters:</p>
+<p>2ul 10x T4 ligase buffer</p>
+<p>3 ul backbone miniprep (6K, 6M and 8A)</p>
+<p>0,5 ul T4 ligase</p>
+<p>0,5 ul BsaI</p>
+<p>1 uL purified PCR</p>
+<p>13 ul H20</p>
+<p>incubate at 37oC for 1h, 16oC 5 min, 55oC 5 min, hold at 4oC</p>
+<p>Transform DH5aplha </p>
+<p>It worked! The MCS with marker was swapped! Successful exchange between MCS of level 2 plasmids 6K, 6M and 8A for MCS pOdd compatible, also exchanging the GFP reporter for the red reporter.</p>
+
+<figure>
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura12.png">
+ </figure>
+
+<p>Golden gate to insert TU1 and TU2 into pOdd1 and pOdd2 (using SapI)</p>
+
+<p>Dry and weigh BC sheets – before drying 14,58 g. After 24 hours at 65oC – 0,157 g</p>
+
+<p>Optimize antibiotic concentrations in HS plates. </p>
+
+<p>Amp 200/300/400 mg/L</p>
+
+<p>Kan 100/150/200/300 mg/L</p>
+
+<p>Chlo 50/100/150/200 mg/L</p>
+
+<p>Spec 50/100/150/200/250 mg/L</p>
+
+
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura13.png">
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura14.png">
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura15.png">
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura16.png">
+ <img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura17.png">
+
+
+<p>Conclusion: the antibiotic concentrations recommended in the literature are excessive for both AF1 and medellinensis. This might explain why are transformations are not working. Even with the plasmids the antibiotic concentration on the plates might still be too high. </p>
+
+<p>test new parameters for electroporation and selection of K. rhaeticus AF1 transformants on plates with lower antibiotic concentration. It WORKS!!!!!!!</p>
+
+<img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura18.png">
+
+<p>culture AF1 for HPLC</p>
+<p>typical task list for the day: </p>
+
+<img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura19.png">
+
+<h1>October 2022: second week </h1>
+
+<p>Transform K. rhaeticus AF1 with the Unicamp_brazil plasmid BBa_K4435305. </p>
+
+<img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura20.png">
+
+
+<p>Goldengate for assembly of CDS, promotor, RBS and terminator into iGEM backbones. Transformations OK. Innoculate 1 colony each for miniprep. Run gel of minipreps (not digested – supercoiled band evident). Candidates for parts BBa_K4435001 to BBa_K4435017:</p>
+<p>LexRO</p>
+<p>Flpe</p>
+<p>dgc1</p>
+<p>galU Kr</p>
+<p>pgm Kr</p>
+<p>ndp Kr</p>
+<p>GK Kr</p>
+<p>galU Km</p>
+<p>pgm Km</p>
+<p>ndp Km</p>
+<p>Gk Km</p>
+<p>18m (original plasmid)</p>
+<p>TUp_a</p>
+<p>TUp_b</p>
+<p>TUp_c</p>
+<p>16c</p>
+
+<img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura21.png">
+
+<p>Conclusion: some promising candidates. Must sequence and inoculate more for minipreps.</p>
+
+<p>Construct parts BBa_K4435306, BBa_K4435307 and BBa_K4435314 (select red colonies).</p>
+
+<img class="model-page-img" src="https://static.igem.wiki/teams/4435/wiki/pages/notebook/figura22.png">
+
+<p>Conclusion: red and green colonies in all plates. Pick 2 red colonies from each plate for miniprep and sequencing. Phenotypically successful. </p>
+
{% endblock %}
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-{% extends "layout.html" %}
-
-{% block title %}Plant{% endblock %}
-{% block lead %}{% endblock %}
-
-{% block page_content %}
-
-<div class="row mt-4">
- <div class="col">
- <div class="bd-callout bd-callout-info">
- <h4>Best Plant Synthetic Biology Special Prize</h4>
- <p>This award is designed to celebrate exemplary work done in plant synthetic biology. Did you build a project in a plant chassis? Did you submit plant parts to the Registry? This award could also be given to a team working with algae or another photosynthetic chassis. Show us what you made and remember to adhere to iGEM safety guidelines!</p>
- <p>To compete for the Best Plant Synthetic Biology prize, please describe your work on this page and also fill out the description on the <a href="https://competition.igem.org/deliverables/judging-form">judging form</a>.</p>
- <hr>
- <p>Please see the <a href="https://competition.igem.org/judging/awards">2022 Awards Page</a> for more information.</p>
- </div>
- </div>
-</div>
-
-<div class="row mt-4">
- <div class="col">
- <h2>Inspirations</h2>
- <hr>
- <ul>
- <li><a href="http://2018.igem.org/Team:Cardiff_Wales/Plant">2018 Cardiff Wales</a></li>
- <li><a href="https://2019.igem.org/Team:Sorbonne_U_Paris/Plant">2019 Sorbonne U Paris</a></li>
- <li><a href="https://2019.igem.org/Team:TU_Kaiserslautern/Plant">2019 TU Kaiserslautern</a></li>
- <li><a href="https://2019.igem.org/Team:Humboldt_Berlin/Plant">2019 Humboldt Berlin</a></li>
- <li><a href="https://2020.igem.org/Team:Sorbonne_U_Paris/Plant">2020 Sorbonne U Paris</a></li>
- </ul>
- </div>
-</div>
-
-{% endblock %}
diff --git a/wiki/pages/partnership.html b/wiki/pages/partnership.html
index 63dfef30f628aa3356a17e6daf0ca36d27ae762c..8e8ecdbfd178019e154b646444705f8999fec170 100644
--- a/wiki/pages/partnership.html
+++ b/wiki/pages/partnership.html
@@ -1,7 +1,7 @@
{% extends "layout.html" %}
{% block title %}Partnership{% endblock %}
-{% block lead %}Collaborate throughout the year with at least one other 2022 iGEM team on a set of shared objectives related to both of your projects.{% endblock %}
+{% block lead %}{% endblock %}
{% block page_content %}
@@ -19,7 +19,7 @@
<p>With a bacterial cellulose production project, by an organism of the same genus as the bacteria used by our team, Ui_Oslo seeks to produce a co-polymer of cellulose and chitin, trough a co-culture assay, that can be used for a large number of applications. Considering our common goals and aiming to contribute to future teams, together we built Celluminati</p>
<h1>Getting to know each other</h1>
-With the dissemination of our project and activities developed on Instagram, Ui_Oslo noticed that our team was also working with bacterial cellulose production and coincidentally with an organism of the same genus (<i>Komagataeibacter sp.</i>). The social network was, therefore, an important means of communication, through which the partner team got in touch with us. It was how we scheduled the first, of many, online meetings to discuss projects, desires, and possible contributions.
+<p>With the dissemination of our project and activities developed on Instagram, Ui_Oslo noticed that our team was also working with bacterial cellulose production and coincidentally with an organism of the same genus (<i>Komagataeibacter sp.</i>). The social network was, therefore, an important means of communication, through which the partner team got in touch with us. It was how we scheduled the first, of many, online meetings to discuss projects, desires, and possible contributions.</p>
<h1>What did team UiO bring to Unicamp?</h1>
<h1>Strategy for dealing with LexRO system </h1>
diff --git a/wiki/pages/protocols.html b/wiki/pages/protocols.html
index 6971d4b680df0cfb18f64aa7c627a18cfee99684..a92836339df8d97f00ce0d2b688e3f9bfa7abee0 100644
--- a/wiki/pages/protocols.html
+++ b/wiki/pages/protocols.html
@@ -7,12 +7,8 @@
<div class="row mt-4">
<div class="col">
- <div class="bd-callout bd-callout-info">
- <h4>Protocols</h4>
- <p>Text.</p>
- <hr>
- <p>Text.</p>
- </div>
+ <h1>Protocols optimised for the Cellulopolis project</h1>
+ <embed src="https://static.igem.wiki/teams/4435/wiki/protocol/protocols.pdf" class="pdf-protocol" type="application/pdf">
</div>
</div>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index c943593f8e10fc759e53869b4107804c7a56e631..9064f6855db1ddfe151f5974fc5789cc1251e864 100644
--- a/wiki/pages/results.html
+++ b/wiki/pages/results.html
@@ -4,199 +4,214 @@
{% block lead %}{% endblock %}
{% block page_content %}
-<style>
- #div-sidenav {
- display: none;
- }
-</style>
-<div class="row mt-4">
- <div class="col">
- <h1>Overview</h1>
- <p>We faced the challenge of developing a project ranging from characterization of non-standard growth media, through BC cultivation, hardware engineering, computer simulation, strain design, plasmid engineering, transformations, and applications of our final product (BC). Due to the broadness of the project and the limited time for its execution, we developed multiple fronts in parallel. Here we present some of the highlights of our work.</p>
+<h1>Overview</h1>
+<p>We faced the challenge of developing a project ranging from characterization of non-standard growth media, through BC cultivation, hardware engineering, computer simulation, strain design, plasmid engineering, transformations, and applications of our final product (BC). Due to the broadness of the project and the limited time for its execution, we developed multiple fronts in parallel. Here we present some of the highlights of our work.</p>
- <div id="results-diagram">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/fluxograma.svg">
- </div>
-
- <h1>Establishment of <i>K. rhaeticus</i> AF1 transformation protocol</h1>
- <p>A consistent transformation protocol is crucial for any strain engineering project. Thus, we invested much work in establishing the ideal conditions for <i>K. rhaeticus</i> AF1 electroporation. Initially, we based our attempts on the strategies used for the transformation of <i>K. rhaeticus</i> iGEM (Goosens, Vivianne J., et al., 2021), however, all of our experiments failed. . Other literature sources were also used in an attempt to carry out the transformation, but none of them resulted in success.</p>
- <p>After troubleshooting parameters such as preparation of competent cells, plasmid selection markers, plasmid replication origins, the intensity of electroporation, cuvette brands, cell density, and many other parameters, we decided to change the suggested antibiotic concentration on our HS agar plates. After all of our frustrating attempts, we finally established a consistent protocol for <i>K. rhaeticus</i> AF1 transformation.</p>
-
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-1.png" >
- <figcaption>Figura 1. Efficiency of <i>Komagataeibacter rhaeticus</i> AF1 tested with different concentrations of antibiotics.</figcaption>
- </figure>
+<div id="results-diagram">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/fluxograma.svg">
+</div>
- <p>Optimized Komagataeibacter rhaeticus AF1 transformation protocol by electroporation (<a href="https://docs.google.com/document/d/1RZtNi85U2spozimZ5CDCeTo409KxxFF08jsHbn2zFRY/edit?usp=sharing" target="_blank">protocol</a>)</p>
+<h1>Establishment of <i>K. rhaeticus</i> AF1 transformation protocol</h1>
+<p>A consistent transformation protocol is crucial for any strain engineering project. Thus, we invested much work in establishing the ideal conditions for <i>K. rhaeticus</i> AF1 electroporation. Initially, we based our attempts on the strategies used for the transformation of <i>K. rhaeticus</i> iGEM (Goosens, Vivianne J., et al., 2021), however, all of our experiments failed. . Other literature sources were also used in an attempt to carry out the transformation, but none of them resulted in success.</p>
+<p>After troubleshooting parameters such as preparation of competent cells, plasmid selection markers, plasmid replication origins, the intensity of electroporation, cuvette brands, cell density, and many other parameters, we decided to change the suggested antibiotic concentration on our HS agar plates. After all of our frustrating attempts, we finally established a consistent protocol for <i>K. rhaeticus</i> AF1 transformation.</p>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-1.png" >
+ <figcaption>Figura 1. Efficiency of <i><i>Komagataeibacter rhaeticus</i></i> AF1 tested with different concentrations of antibiotics.</figcaption>
+</figure>
- <h1>Toolbox</h1>
- <h2>Construction of broad species spectrum plasmids with compatible selection markers</h2>
- (further details in the parts section)
+<p>Optimized <i>Komagataeibacter rhaeticus</i> AF1 transformation protocol by electroporation (<a href="https://docs.google.com/document/d/1RZtNi85U2spozimZ5CDCeTo409KxxFF08jsHbn2zFRY/edit?usp=sharing" target="_blank">protocol</a>)</p>
- <p>Komagataeibacter is a non-conventional chassis in which traditional E. coli replication origins are not functional. Hence, plasmid replication requires broad-spectrum origins such as RK2, pBBR1 or RSF1010. Within iGEM´s 2022 distribution kit there were 6 plasmids with such requirements, namely the GoldenGate level 1: BBa_J428346, BBa_J428347, and BBa_J428349 (KanR), and the GoldenGate level 2: BBa_J428366, BBa_J428367, and BBa_J428369 (SpecR). This posed a potential problem to our design strategy as, to the best of our knowledge, there is no report of successful use of the SpecR marker in K. rhaeticus. Thus, we design a strategy to replace the KanR and SpecR marker from level 1 and 2 plasmids (respectively) with both AmpR and CmR. This was accomplished by designing the primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers (for details, see parts section). In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357. Employing digestion with SapI and ligation with T4 DNA ligase, we succeeded in assembling new level 1 and level 2 Komagateibacter compatible plasmids with different marker options.</p>
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-2.png"
- class="figures-left">
- <figcaption>Figure 2. Amplification of plasmid backbones with JUMP-mF and JUMP-mR: A.BBa_J428346, B.BBa_J428347, C.BBa_J428367, D.BBa_J428349, E.BBa_J428366 and F.BBa_J428369.</figcaption>
- </figure>
+<h1>Toolbox</h1>
+<h2>Construction of broad species spectrum plasmids with compatible selection markers</h2>
+(further details in the parts section)
+
+<p>Komagataeibacter is a non-conventional chassis in which traditional <i>E. coli</i> replication origins are not functional. Hence, plasmid replication requires broad-spectrum origins such as RK2, pBBR1 or RSF1010. Within iGEM´s 2022 distribution kit there were 6 plasmids with such requirements, namely the GoldenGate level 1: BBa_J428346, BBa_J428347, and BBa_J428349 (KanR), and the GoldenGate level 2: BBa_J428366, BBa_J428367, and BBa_J428369 (SpecR). This posed a potential problem to our design strategy as, to the best of our knowledge, there is no report of successful use of the SpecR marker in <i>K. rhaeticus</i>. Thus, we design a strategy to replace the KanR and SpecR marker from level 1 and 2 plasmids (respectively) with both AmpR and CmR. This was accomplished by designing the primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers (for details, see parts section). In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357. Employing digestion with SapI and ligation with T4 DNA ligase, we succeeded in assembling new level 1 and level 2 Komagateibacter compatible plasmids with different marker options.</p>
+
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-2.png">
+ <figcaption>Figure 2. Amplification of plasmid backbones with JUMP-mF and JUMP-mR: A.BBa_J428346, B.BBa_J428347, C.BBa_J428367, D.BBa_J428349, E.BBa_J428366 and F.BBa_J428369.</figcaption>
+</figure>
+
+<p>Thus, we succeeded in constructing the following plasmids:</p>
- <p>Thus, we succeeded in constructing the following plasmids:</p>
+<table>
+ <tr><th>New backbones</th><th>Details</th><th>Internal Code</th><th>Validation</th></tr>
+ <tr><td>BBa_K4435301</td><td>amplification of BBa_J428346 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2K to AmpR</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435302</td><td>amplification of BBa_J428347 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2M to AmpR</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435303</td><td>amplification of BBa_J428349 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>4A to AmpR</td><td></td></tr>
+ <tr><td>BBa_K4435304</td><td>amplification of BBa_J428366 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6K to AmpR</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435305</td><td>amplification of BBa_J428367 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6M to AmpR</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435308</td><td>amplification of BBa_J428346 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2K to CmR</td><td></td></tr>
+ <tr><td>BBa_K4435309</td><td>amplification of BBa_J428347 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2M to CmR</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435310</td><td>amplification of BBa_J428349 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>4A to CmR</td><td></td></tr>
+ <tr><td>BBa_K4435311</td><td>amplification of BBa_J428366 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6K to CmR</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435312</td><td>amplification of BBa_J428367 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6M to CmR</td><td></td></tr>
+ <tr><td>BBa_K4435315</td><td>add ODD 4 TUs adapter, derived from BBa_J428366: pJUMP42x-2A SpecR Type IIS Level 2 vector. Origin RK2 (broad-host-range);</td><td>6K_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435316</td><td>add ODD 4 TUs adapter, derived from BBa_J428367: pJUMP43-2A SpecR Type IIS Level 2 vector. Origin pBBR1 (medium-copy, broad-host-range)</td><td>6M_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435317</td><td>add ODD 4 TUs adapter, derived from BBa_J428369: pJUMP45-2A SpecR Type IIS Level 2 vector. Origin RSF1010 (Broad-host-range)</td><td>8A_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435306</td><td>add ODD 4 TUs adapter to BBa_K4435304</td><td>6K to AmpR_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435307</td><td>add ODD 4 TUs adapter to BBa_K4435305</td><td>6M to AmpR_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435313</td><td>add ODD 4 TUs adapter to BBa_K4435311</td><td>6K to CmR_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435314</td><td>add ODD 4 TUs adapter to BBa_K4435312</td><td>6M to CmR_red</td><td></td></tr>
+</table>
- <table>
- <tr><th>New backbones</th><th>Details</th><th>Internal Code</th><th>Validation</th></tr>
-<tr><td>BBa_K4435301</td><td>amplification of BBa_J428346 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2K to AmpR</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435302</td><td>amplification of BBa_J428347 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2M to AmpR</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435303</td><td>amplification of BBa_J428349 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>4A to AmpR</td><td></td></tr>
-<tr><td>BBa_K4435304</td><td>amplification of BBa_J428366 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6K to AmpR</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435305</td><td>amplification of BBa_J428367 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6M to AmpR</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435308</td><td>amplification of BBa_J428346 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2K to CmR</td><td></td></tr>
-<tr><td>BBa_K4435309</td><td>amplification of BBa_J428347 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>2M to CmR</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435310</td><td>amplification of BBa_J428349 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>4A to CmR</td><td></td></tr>
-<tr><td>BBa_K4435311</td><td>amplification of BBa_J428366 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6K to CmR</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435312</td><td>amplification of BBa_J428367 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI</td><td>6M to CmR</td><td></td></tr>
-<tr><td>BBa_K4435315</td><td>add ODD 4 TUs adapter, derived from BBa_J428366: pJUMP42x-2A SpecR Type IIS Level 2 vector. Origin RK2 (broad-host-range);</td><td>6K_red</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435316</td><td>add ODD 4 TUs adapter, derived from BBa_J428367: pJUMP43-2A SpecR Type IIS Level 2 vector. Origin pBBR1 (medium-copy, broad-host-range)</td><td>6M_red</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435317</td><td>add ODD 4 TUs adapter, derived from BBa_J428369: pJUMP45-2A SpecR Type IIS Level 2 vector. Origin RSF1010 (Broad-host-range)</td><td>8A_red</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435306</td><td>add ODD 4 TUs adapter to BBa_K4435304</td><td>6K to AmpR_red</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435307</td><td>add ODD 4 TUs adapter to BBa_K4435305</td><td>6M to AmpR_red</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435313</td><td>add ODD 4 TUs adapter to BBa_K4435311</td><td>6K to CmR_red</td><td>phenotype</td></tr>
-<tr><td>BBa_K4435314</td><td>add ODD 4 TUs adapter to BBa_K4435312</td><td>6M to CmR_red</td><td></td></tr>
- </table>
+<p>Phenotipic validation: Image illustrating <i>E. coli</i> strains harbouring novel plasmids and streaked onto plates with the indicated antibiotics.</p>
- <p>Phenotipic validation: Image illustrating E. coli strains harbouring novel plasmids and streaked onto plates with the indicated antibiotics.</p>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-8.png">
+ <figcaption>Figure 3. <i>E. coli</i> transformed with swapped markers plasmids growing in media containing antibiotics.</figcaption>
+</figure>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-8.png">
- <figcaption>Figure 3. E. coli transformed with swapped markers plasmids growing in media containing antibiotics.</figcaption>
- </figure>
+<p>Our final proof of success is the transformation of Komagataeibacter with part BBa_K4435305, which has the new antibiotic resistance cassette and shows it’s efficiency in <i>Komagataeibacter rhaeticus</i> AF1 (5 and 10 mg/L ampicillin allowed background growth of cells electroporated but without plasmids; 400 mg/L - the recommended ampicillin concentration in the literature - exceeded the tolerable levels for the AF1 strain; 25 mg/L of ampicillin did not allow the growth of cells without plasmids but selected positive transformants). All the new plasmids with swapped markers are await for sequencing.</p>
- <p>Our final proof of success is the transformation of Komagataeibacter with part BBa_K4435305, which has the new antibiotic resistance cassette and shows it’s efficiency in Komagataeibacter rhaeticus AF1 (5 and 10 mg/L ampicillin allowed background growth of cells electroporated but without plasmids; 400 mg/L - the recommended ampicillin concentration in the literature - exceeded the tolerable levels for the AF1 strain; 25 mg/L of ampicillin did not allow the growth of cells without plasmids but selected positive transformants). All the new plasmids with swapped markers are await for sequencing.</p>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-3.png">
+ <figcaption>Figure 4. Different concentrations of antibiotics used in bacteria transformation.</figcaption>
+</figure>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-3.png">
- <figcaption>Figure 4. Different concentrations of antibiotics used in bacteria transformation.</figcaption>
- </figure>
+<p>Our engineering strategy for the replacement of bcsZ or bcsA promotors requires the genomic integration of cassettes with include: 1 kb of homology to the region 5´the integration locus, recyclable antibiotic resistance marker (AmpR flanked by FRT recombination sites), transcriptional unit encoding the LexRO transcriptional repressor under the control of strong to medium promotors, and a final module encoding the LexRO binding region (operator) followed by RBS and 1 kb of homology to the region 3´the integration locus. This design requires level 2 assembly of 4 transcriptional units (basic TUs assembled in pOdd1 to pOdd4 plasmids).</p>
- <p>Our engineering strategy for the replacement of bcsZ or bcsA promotors requires the genomic integration of cassettes with include: 1 kb of homology to the region 5´the integration locus, recyclable antibiotic resistance marker (AmpR flanked by FRT recombination sites), transcriptional unit encoding the LexRO transcriptional repressor under the control of strong to medium promotors, and a final module encoding the LexRO binding region (operator) followed by RBS and 1 kb of homology to the region 3´the integration locus. This design requires level 2 assembly of 4 transcriptional units (basic TUs assembled in pOdd1 to pOdd4 plasmids).</p>
+<p>In parallel, we also cloned in level 0 plasmids genes (from <i>K. rhaeticus</i> and <i>K. medellinensis</i>) encoding key proteins in the pathway that converts glucose to BC (awaiting sequencing results). These will be assembled into transcriptional units TU1 to TU4 in pOdd1 to pOdd4 plasmids. Concomitant overexpression of 4 different proteins requires their assembly into level 2 <i>Komagataeibacter</i> compatible plasmids.</p>
- <p>In parallel, we also cloned in level 0 plasmids genes (from K. rhaeticus and K. medellinensis) encoding key proteins in the pathway that converts glucose to BC (awaiting sequencing results). These will be assembled into transcriptional units TU1 to TU4 in pOdd1 to pOdd4 plasmids. Concomitant overexpression of 4 different proteins requires their assembly into level 2 <i>Komagataeibacter</i> compatible plasmids.</p>
+<p>Unfortunately, the available backbones did not meet our needs, which requires the presence of SapI restriction with ends complementary to 5´of TU1 (pOdd1) and 3´of TU4 (pOdd4), thus we designed primers AdapterTU_reD_F and AdapterTU_reD_R and amplified the red reporter from BBa_J04452. PCR products were assembled by GoldenGate using BsaI and T4 DNA ligase with level 2 plasmids. Successful assembly yielded red colonies and original unedited plasmids yielded green colonies.</p>
- <p>Unfortunately, the available backbones did not meet our needs, which requires the presence of SapI restriction with ends complementary to 5´of TU1 (pOdd1) and 3´of TU4 (pOdd4), thus we designed primers AdapterTU_reD_F and AdapterTU_reD_R and amplified the red reporter from BBa_J04452. PCR products were assembled by GoldenGate using BsaI and T4 DNA ligase with level 2 plasmids. Successful assembly yielded red colonies and original unedited plasmids yielded green colonies.</p>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/registry-parts/level-2-red-adapter-colonies.png">
+ <figcaption>Figure 5. LB Amp plates with green colonies indicating the transformation with the original cloning site (replacement not sucessfull), and red colonies showing the sucessfull introduction of pOdd1-4 compatible level 2 adaptor.</figcaption>
+</figure>
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-4.png" class="figures-left">
- <figcaption>Figure 5. LB Amp plates with green colonies indicating the transformation with the original cloning site (replacement not sucessfull), and red colonies showing the sucessfull introduction of pOdd1-4 compatible level 2 adaptor.</figcaption>
- </figure>
+<p>New backbones (Type IIs compatible):</p>
- <p>New backbones (Type IIs compatible):</p>
+<table>
+ <tr><th>New backbones</th><th>Source</th><th>Internal Code</th><th>Validation</th></tr>
+ <tr><td>BBa_K4435315</td><td>add ODD 4 TUs adapter, derived from BBa_J428366: pJUMP42x-2A SpecR Type IIS Level 2 vector. Origin RK2 (broad-host-range)</td><td>6K_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435316</td><td>add ODD 4 TUs adapter, derived from BBa_J428367: pJUMP43-2A SpecR Type IIS Level 2 vector. Origin pBBR1 (medium-copy, broad-host-range)</td><td>6M_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435317</td><td>add ODD 4 TUs adapter, derived from BBa_J428369: pJUMP45-2A SpecR Type IIS Level 2 vector. Origin RSF1010 (Broad-host-range)</td><td>8A_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435306</td><td>add ODD 4 TUs adapter to BBa_K4435304</td><td>6K to AmpR_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435307</td><td>add ODD 4 TUs adapter to BBa_K4435305</td><td>6M to AmpR_red</td><td>phenotype</td></tr>
+ <tr><td>BBa_K4435313</td><td>add ODD 4 TUs adapter to BBa_K4435311</td><td>6K to CmR_red</td><td>under construction</td></tr>
+ <tr><td>BBa_K4435314</td><td>add ODD 4 TUs adapter to BBa_K4435312</td><td>6M to CmR_red</td><td>phenotype</td></tr>
+</table>
- <p>Table 2</p>
+<h2>Cloning the basic parts</h2>
- <h2>Cloning the basic parts</h2>
+<p>Considering the BC synthesis pathway we were able to identify key enzymes involved in cellulose production, specially when Glucose is the main carbon source for this biosynthesis. Trough our modeling, we confirm which main enzymes could be overexpressed and help in the production of cellulose.</p>
+<p>After glucose (GLC) import we see this conversion into D-glucose-6-phosphate (G6P) by glucokinase (GK), which is then converted into D-glucose-1-phosphate (G1P) by UTP-glucose-1-phosphate uridylyltransferase (galU). From that UDP-glucose (UDPG) is produced by hosphoglucomutase (pgm), which requires UTP by nucleoside diphosphate pyrophosphorylase (ndp) production. UDPG works as a substrate for cellulose synthesis by bcsA, which requires cyclic diguanylic acid (C-di-GMP) produced by diguanylate cyclase (dgc).</p>
- <p>Considering the BC synthesis pathway we were able to identify key enzymes involved in cellulose production, specially when Glucose is the main carbon source for this biosynthesis. Trough our modeling, we confirm which main enzymes could be overexpressed and help in the production of cellulose.</p>
- <p>After glucose (GLC) import we see this conversion into D-glucose-6-phosphate (G6P) by glucokinase (GK), which is then converted into D-glucose-1-phosphate (G1P) by UTP-glucose-1-phosphate uridylyltransferase (galU). From that UDP-glucose (UDPG) is produced by hosphoglucomutase (pgm), which requires UTP by nucleoside diphosphate pyrophosphorylase (ndp) production. UDPG works as a substrate for cellulose synthesis by bcsA, which requires cyclic diguanylic acid (C-di-GMP) produced by diguanylate cyclase (dgc).</p>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-5.png">
+ <figcaption>Figure 6. Bacterial cellulose production pathway. Adapted from <a href="https://doi.org/10.4014/jmb.2006.06026">Hur et al., 2020</a></figcaption>
+</figure>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-5.png">
- <figcaption>Figure 6. Bacterial cellulose production pathway. Adapted from <a href="https://doi.org/10.4014/jmb.2006.06026">Hur et al., 2020</a></figcaption>
- </figure>
+<p>In order to obtain experimental results consistents with the modelling prediction, identifying the limiting enzyme in the pathway, we amplified genes encoding GK, galU, pgm and ndp from genomic DNA of <i>K. rhaeticus</i> AF1 and <i>K. medellinensis</i>, along with the synthesis of dgcI. These are being cloned into BBa_J428381, BBa_J428382, BBa_J428383, and BBa_J428384, under the control of the strong constitutive promotor BBa_K4435012, RBS BBa_K4435015 and terminator BBa_K4435017 (given us level 1 composite parts BBa_K4435120 to BBa_K4435127).</p>
- <p>In order to obtain experimental results consistents with the modelling prediction, identifying the limiting enzyme in the pathway, we amplified genes encoding GK, galU, pgm and ndp from genomic DNA of K. rhaeticus AF1 and K. medellinensis, along with the synthesis of dgcI. These are being cloned into BBa_J428381, BBa_J428382, BBa_J428383, and BBa_J428384, under the control of the strong constitutive promotor BBa_K4435012, RBS BBa_K4435015 and terminator BBa_K4435017 (given us level 1 composite parts BBa_K4435120 to BBa_K4435127).</p>
+<p>The constructs were transformed into <i>E. coli</i>, which grew efficiently, proving the antibiotic resistance and, consequently, the right assembly of the plasmids. Some of it’s resistant colonies were isolated, plasmid prepared, and analyzed by agarose gel electrophoresis. The band sizes show many candidates were sent for sequencing. Further colonies are being selected for constructs where we have not selected plasmids of the correct size in our first attempt (only 1 colony was selected per plate in our first screen). We are confident that we have level 0 plasmids for all our basic parts (awaits confirmation).</p>
- <p>The constructs were transformed into E. coli, which grew efficiently, proving the antibiotic resistance and, consequently, the right assembly of the plasmids. Some of it’s resistant colonies were isolated, plasmid prepared, and analyzed by agarose gel electrophoresis. The band sizes show many candidates were sent for sequencing. Further colonies are being selected for constructs where we have not selected plasmids of the correct size in our first attempt (only 1 colony was selected per plate in our first screen). We are confident that we have level 0 plasmids for all our basic parts (awaits confirmation).</p>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-6.png">
+ <figcaption>Figure 7. Example of gel electrophoresis of undigested (supercoilled) plasmids with candidate level 0 parts.</figcaption>
+</figure>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-6.png">
- <figcaption>Figure 7. Example of gel electrophoresis of undigested (supercoilled) plasmids with candidate level 0 parts.</figcaption>
- </figure>
+<p>Parts listed below will be assembled into transcriptional units (TU1 to TU4) in pOdd 1 to 4 plasmids for further level 2 assembly into BBa_K4435306 or BBa_K4435307. These can then be transformed into <i>K. rhaeticus</i> and BC production yields are quantified.</p>
- <p>Parts listed below will be assembled into transcriptional units (TU1 to TU4) in pOdd 1 to 4 plasmids for further level 2 assembly into BBa_K4435306 or BBa_K4435307. These can then be transformed into K. rhaeticus and BC production yields are quantified.</p>
+<p>Level 0 (Type IIs compatible):</p>
- <h1>Hardware</h1>
- <h2>Hardware - dark vs light</h2>
+<table>
+ <tr><th>Level 0 Part</th><th>Short name</th><th>Origin and protein cloned</th></tr>
+ <tr><td>BBa_K4435004</td><td>Kr_galU_TU1</td><td><i>K. rhaeticus</i> AF1 galU_UTP-glucose-1-phosphate uridylyltransferase</td></tr>
+ <tr><td>BBa_K4435005</td><td>Kr_pgm_TU2</td><td><i>K. rhaeticus</i> AF1 pgm_Phosphoglucomutase</td></tr>
+ <tr><td>BBa_K4435006</td><td>Kr_ndp_TU3</td><td><i>K. rhaeticus</i> AF1 ndp_Nucleoside diphosphate pyrophosphorylase</td></tr>
+ <tr><td>BBa_K4435007</td><td>Kr_GK_TU4</td><td><i>K. rhaeticus</i> AF1 GK_Glucokinase</td></tr>
+ <tr><td>BBa_K4435008</td><td>Km_galU_TU1</td><td><i>K. medellinensis</i> galU_UTP-glucose-1-phosphate uridylyltransferase</td></tr>
+ <tr><td>BBa_K4435009</td><td>Km_pgm_TU2</td><td><i>K. medellinensis</i> pgm_Phosphoglucomutase</td></tr>
+ <tr><td>BBa_K4435010</td><td>Km_ndp_TU3</td><td><i>K. medellinensis</i> ndp_Nucleoside diphosphate pyrophosphorylase</td></tr>
+ <tr><td>BBa_K4435011</td><td>Km_GK_TU4</td><td><i>K. medellinensis</i> GK_Glucokinase</td></tr>
+ <tr><td>BBa_K4435003</td><td>dgc1_TU4</td><td>dgc1_diguanylate cyclase</td></tr>
+</table>
- <p>Aiming to improve our cellulose production and detach dark (no BC production) and light (bcs induction) phases of the process, we designed and built a two step bioreactor. As our project proposes, Komagataeibacter rhaeticus AF1 should have a period of growth, in the absence of light, followed by activation of it’s bcs operon by luminous stimulus. To predict the best moment to start the induction of cellulose production, resulting in a higher amount of final product, we comply our math model. The conditions of presence or privation of light and even the change-over them can be respected in the developed hardware. Experimental tests demonstrate the the efficiency of bioreactor by the exponencial growth of E. coli and suggested a big condition advantage for obligate aerobic organisms.</p>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-9.png">
- <figcaption>Figure 8. Bioreactor produced in the lab.</figcaption>
- </figure>
+<h1>Hardware</h1>
+<h2>Hardware - dark vs light</h2>
- <p>The operation of the bioreactor is simple, it works with (a) the culture medium with genetically modified KAF1 where as soon as it reaches a maximum OD, without harming the colony, it will be inserted into our closed system (b) through a hose that connects (a) and (b). And thus we activate the blue light that is contained in its lid. In this way we can fully control the cellulose production. All materials in the bioreactor are sterile.</p>
+<p>Aiming to improve our cellulose production and detach dark (no BC production) and light (bcs induction) phases of the process, we designed and built a two step bioreactor. As our project proposes, <i>Komagataeibacter rhaeticus</i> AF1 should have a period of growth, in the absence of light, followed by activation of it’s bcs operon by luminous stimulus. To predict the best moment to start the induction of cellulose production, resulting in a higher amount of final product, we comply our math model. The conditions of presence or privation of light and even the change-over them can be respected in the developed hardware. Experimental tests demonstrate the the efficiency of bioreactor by the exponencial growth of <i>E. coli</i> and suggested a big condition advantage for obligate aerobic organisms.</p>
- <h1>Glucose consumption by Komagataeibacter rhaeticus AF1</h1>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-9.png">
+ <figcaption>Figure 8. Bioreactor produced in the lab.</figcaption>
+</figure>
- <h2>HS medium vs orange juice</h2>
+<p>The operation of the bioreactor is simple, it works with (a) the culture medium with genetically modified KAF1 where as soon as it reaches a maximum OD, without harming the colony, it will be inserted into our closed system (b) through a hose that connects (a) and (b). And thus we activate the blue light that is contained in its lid. In this way we can fully control the cellulose production. All materials in the bioreactor are sterile.</p>
- <h3>1. standard media (HS)</h3>
+<h1>Glucose consumption by <i>Komagataeibacter rhaeticus</i> AF1</h1>
- <p>Komagataeibacter was grown in liquid HS media, which has D-glucose as its sugar source. We asked ourselves if the bacteria use this glucose as a substrate for cellulose production. So, in order to evaluate glucose consumption, we used a quantification technique based on High Performance Liquid Chromatography (HPLC).</p>
+<h2>HS medium vs orange juice</h2>
- <p>For this analysis, we injected 5 samples collected from different days of bacterial growth, to compare their sugar elution profile to a standard sample of monosaccharides and short-chain carbohydrates. This way it was possible to identify and quantify the sugars present in our sample by integrating their corresponding peaks.</p>
+<h3>1. standard media (HS)</h3>
- <p>The media samples’ elution profiles showed peaks that matched with glucose and, the short-chain sugar made of three units of glucose, cellotriose. It is possible to see glucose’s concentration decrease, as well as the cellotriose’s concentration increase as the growth days go by in the chromatograms. This observation shows that the glucose monomer is consumed by the bacteria and glucose polymers are being formed, as glucose decrease coincides with cellotriose’s increase (Figure 1).</p>
+<p>Komagataeibacter was grown in liquid HS media, which has D-glucose as its sugar source. We asked ourselves if the bacteria use this glucose as a substrate for cellulose production. So, in order to evaluate glucose consumption, we used a quantification technique based on High Performance Liquid Chromatography (HPLC).</p>
- <p>The chromatograms obtained are available <a href="https://static.igem.wiki/teams/4435/wiki/pages/results/carbohydrate-quantification-protocol-1.pdf">here</a></p>
+<p>For this analysis, we injected 5 samples collected from different days of bacterial growth, to compare their sugar elution profile to a standard sample of monosaccharides and short-chain carbohydrates. This way it was possible to identify and quantify the sugars present in our sample by integrating their corresponding peaks.</p>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/carbohydratequantification.svg">
- <figcaption>Figure 9. Glucose and cellotriose concentrations through the days of Komagataeibacter’s growth in HS liquid media.</figcaption>
- </figure>
+<p>The media samples’ elution profiles showed peaks that matched with glucose and, the short-chain sugar made of three units of glucose, cellotriose. It is possible to see glucose’s concentration decrease, as well as the cellotriose’s concentration increase as the growth days go by in the chromatograms. This observation shows that the glucose monomer is consumed by the bacteria and glucose polymers are being formed, as glucose decrease coincides with cellotriose’s increase (Figure 1).</p>
+<p>The chromatograms obtained are available <a href="https://static.igem.wiki/teams/4435/wiki/pages/results/carbohydrate-quantification-protocol-1.pdf">here</a></p>
- <h3>2. orange residue media</h3>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/carbohydratequantification.svg">
+ <figcaption>Figure 9. Glucose and cellotriose concentrations through the days of Komagataeibacter’s growth in HS liquid media.</figcaption>
+</figure>
- <p>We also wondered about glucose concentration in the alternative orange residue media, in a way to predict bacteria growth. So we measured it in the HPLC as well. The chromatogram in figure shows a concentration of 3.1836 g/L of glucose and the presence of other carbohydrates that were not identified, as they are not in the standard sample of monosaccharides and short-chain carbohydrates.</p>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/glucosequantificationorangemedia.svg">
- <figcaption>Figure 10. Orange residues media chromatogram for sugar quantification.</figcaption>
- </figure>
+<h3>2. orange residue media</h3>
- <h1>BC production in defined shapes</h1>
- <h2>3D structures</h2>
- <p>With the goal of producing BC membranes that could coat complex 3-dimensional structures, we investigated the requirements for unfolding the surface of different objects onto 2D. By using the perimeter of this 2D object as a guide, we devised a strategy to 3D print molds for the production of BC sheets. As proof of principle, we constructed molds representing the iGEM logo and the surface of a sphere and used to produce BC sheets.</p>
+<p>We also wondered about glucose concentration in the alternative orange residue media, in a way to predict bacteria growth. So we measured it in the HPLC as well. The chromatogram in figure shows a concentration of 3.1836 g/L of glucose and the presence of other carbohydrates that were not identified, as they are not in the standard sample of monosaccharides and short-chain carbohydrates.</p>
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-11.png" class="implementation-img">
- <figcaption>Figure 11. Cellulose grow in 3D molds for cell cultivation</figcaption>
- <hr>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/glucosequantificationorangemedia.svg">
+ <figcaption>Figure 10. Orange residues media chromatogram for sugar quantification.</figcaption>
+</figure>
+<h1>BC production in defined shapes</h1>
+<h2>3D structures</h2>
+<p>With the goal of producing BC membranes that could coat complex 3-dimensional structures, we investigated the requirements for unfolding the surface of different objects onto 2D. By using the perimeter of this 2D object as a guide, we devised a strategy to 3D print molds for the production of BC sheets. As proof of principle, we constructed molds representing the iGEM logo and the surface of a sphere and used to produce BC sheets.</p>
- <h2>Scaffold for cell culture</h2>
+<img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-11.png" class="implementation-img">
+<figcaption>Figure 11. Cellulose grow in 3D molds for cell cultivation</figcaption>
+<hr>
- <p>An exceptional application of BC is its use as a scaffold for cell culture, once it forms colloidal level dispersions in an aqueous medium and shows strong and crystalline structure, boosting cell culturing experiments.(Madhushree Bhattachary et al., 2012). In previous studies, bacterial cellulose has already been tested and shown to be convenient as a scaffold for hard tissues (eg. bone and cartilage). Aiming to evaluate the adherence of distinct cell lines in BC and their efficiency as scaffolds we perform a vast number of assays during our project. First of all, we used the lineage of human skin fibroblast NIH3T3 and reached a praiseworthy cell adhesion in all the groups. In the figure below we can see cells under brightfield microscopy, with a morphology slightly different from the expected pattern, but adhered to the blanket as expected.</p>
+<h2>Scaffold for cell culture</h2>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-7.png">
- <figcaption>Figure 12. Fibroblast adherence in cellulose sheet colored with DAP (blue for nucleus) and Phalloidin (green for actin) revealling it`s morphology</figcaption>
- </figure>
+<p>An exceptional application of BC is its use as a scaffold for cell culture, once it forms colloidal level dispersions in an aqueous medium and shows strong and crystalline structure, boosting cell culturing experiments.(Madhushree Bhattachary et al., 2012). In previous studies, bacterial cellulose has already been tested and shown to be convenient as a scaffold for hard tissues (eg. bone and cartilage). Aiming to evaluate the adherence of distinct cell lines in BC and their efficiency as scaffolds we perform a vast number of assays during our project. First of all, we used the lineage of human skin fibroblast NIH3T3 and reached a praiseworthy cell adhesion in all the groups. In the figure below we can see cells under brightfield microscopy, with a morphology slightly different from the expected pattern, but adhered to the blanket as expected.</p>
- <p>The second batch of tests was with the SK-MEL-28 strain of human melanoma, in which we obtained good adhesion to bacterial cellulose, however, morphologically, they do not appear to undergo cell differentiation, since they have a phenotype similar to that found when the cell does not is adhered to the substrate. In addition to these points, we could also observe that cell adhesion is greater in thinner cellulose when compared to thicker cellulose.</p>
- <p>In view of the results obtained, we were able to identify some points that are amenable to the improvement of our design of experiment, as a way of standardizing the blankets used for cultivation, minimizing the effects of variable parameters. In this scenario, we rethink our design and proposed the use of a 3D mold (<a href="{{ url_for('pages', page='hardware') }}">hardware</a>) that allows the standardization of BC production in optimal size and thickness for cell culture. After the growth of these new cellulose sheets, we ran an assay with C2c12 mouse myoblasts, which shows the efficiency of cell adherence and differentiation rate. The standardization of the sheets seems to be an effective improvement for cell culture as we can see from the pictures. Some observations allowed during these experiments propose the requirement of thinner sheets.</p>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-7.png">
+ <figcaption>Figure 12. Fibroblast adherence in cellulose sheet colored with DAP (blue for nucleus) and Phalloidin (green for actin) revealling it`s morphology</figcaption>
+</figure>
- <figure class="figures-left">
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-12.png">
- <figcaption>Figure 13. Myoblasts adhesion in cellulose sheets</figcaption>
- </figure>
+<p>The second batch of tests was with the SK-MEL-28 strain of human melanoma, in which we obtained good adhesion to bacterial cellulose, however, morphologically, they do not appear to undergo cell differentiation, since they have a phenotype similar to that found when the cell does not is adhered to the substrate. In addition to these points, we could also observe that cell adhesion is greater in thinner cellulose when compared to thicker cellulose.</p>
- <h1>References</h1>
- <ul>
- <li>Helander, Anders, Asgeir Husa, and Jan-Olof Jeppsson. "Improved HPLC method for carbohydrate-deficient transferrin in serum." Clinical chemistry 49.11 (2003): 1881-1890.</li>
- <li>Muir, Jane G., et al. "Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)." Journal of agricultural and food chemistry 57.2 (2009): 554-565.</li>
- <li>Helander, Anders, Asgeir Husa, and Jan-Olof Jeppsson. "Improved HPLC method for carbohydrate-deficient transferrin in serum." Clinical chemistry 49.11 (2003): 1881-1890.</li>
- <li>Muir, Jane G., et al. "Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)." Journal of agricultural and food chemistry 57.2 (2009): 554-565.</li>
- <li>Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).
- Hur DH, Choi WS, Kim TY, Lee SY, Park* JH, Jeong* KJ. Enhanced Production of Bacterial Cellulose in Komagataeibacter xylinus Via Tuning of Biosynthesis Genes with Synthetic RBS. J. Microbiol. Biotechnol. 2020;30:1430-1435.</li>
- <li>Madhushree Bhattacharya, Melina M. Malinen, Patrick Lauren, Yan-Ru Lou, Saara W. Kuisma, Liisa Kanninen, Martina Lille, Anne Corlu, Christiane GuGuen-Guillouzo, Olli Ikkala, Antti Laukkanen, Arto Urtti, Marjo Yliperttula, Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture, Journal of Controlled Release, Volume 164, Issue 3, 2012,Pages 291-298</li>
- <li>Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria Vivianne J. Goosens, Kenneth T. Walker, Silvia M. Aragon, Amritpal Singh, Vivek R. Senthivel, Linda Dekker, Joaquin Caro-Astorga, Marianne L. A. Buat, Wenzhe Song, Koon-Yang Lee, and Tom Ellis ACS Synthetic Biology 2021 10 </li>
+<p>In view of the results obtained, we were able to identify some points that are amenable to the improvement of our design of experiment, as a way of standardizing the blankets used for cultivation, minimizing the effects of variable parameters. In this scenario, we rethink our design and proposed the use of a 3D mold (<a href="{{ url_for('pages', page='hardware') }}">hardware</a>) that allows the standardization of BC production in optimal size and thickness for cell culture. After the growth of these new cellulose sheets, we ran an assay with C2c12 mouse myoblasts, which shows the efficiency of cell adherence and differentiation rate. The standardization of the sheets seems to be an effective improvement for cell culture as we can see from the pictures. Some observations allowed during these experiments propose the requirement of thinner sheets.</p>
- </ul>
- </div>
-</div>
+<figure class="figures-left">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-12.png">
+ <figcaption>Figure 13. Myoblasts adhesion in cellulose sheets</figcaption>
+</figure>
+<h1>References</h1>
+<ul>
+ <li>Helander, Anders, Asgeir Husa, and Jan-Olof Jeppsson. "Improved HPLC method for carbohydrate-deficient transferrin in serum." Clinical chemistry 49.11 (2003): 1881-1890.</li>
+ <li>Muir, Jane G., et al. "Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)." Journal of agricultural and food chemistry 57.2 (2009): 554-565.</li>
+ <li>Helander, Anders, Asgeir Husa, and Jan-Olof Jeppsson. "Improved HPLC method for carbohydrate-deficient transferrin in serum." Clinical chemistry 49.11 (2003): 1881-1890.</li>
+ <li>Muir, Jane G., et al. "Measurement of short-chain carbohydrates in common Australian vegetables and fruits by high-performance liquid chromatography (HPLC)." Journal of agricultural and food chemistry 57.2 (2009): 554-565.</li>
+ <li>Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).</li>
+ <li>Hur DH, Choi WS, Kim TY, Lee SY, Park* JH, Jeong* KJ. Enhanced Production of Bacterial Cellulose in <i>Komagataeibacter xylinus</i> via Tuning of Biosynthesis Genes with Synthetic RBS. J. Microbiol. Biotechnol. 2020;30:1430-1435.</li>
+ <li>Madhushree Bhattacharya, Melina M. Malinen, Patrick Lauren, Yan-Ru Lou, Saara W. Kuisma, Liisa Kanninen, Martina Lille, Anne Corlu, Christiane GuGuen-Guillouzo, Olli Ikkala, Antti Laukkanen, Arto Urtti, Marjo Yliperttula, Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture, Journal of Controlled Release, Volume 164, Issue 3, 2012,Pages 291-298</li>
+ <li>Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria Vivianne J. Goosens, Kenneth T. Walker, Silvia M. Aragon, Amritpal Singh, Vivek R. Senthivel, Linda Dekker, Joaquin Caro-Astorga, Marianne L. A. Buat, Wenzhe Song, Koon-Yang Lee, and Tom Ellis ACS Synthetic Biology 2021 10 </li>
+</ul>
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diff --git a/wiki/pages/safety.html b/wiki/pages/safety.html
index d326f8720954c2562e2eb4a7f444a2a90044f416..1451981f79295f1a9ecf52b97da1f41ffb8ef2b2 100644
--- a/wiki/pages/safety.html
+++ b/wiki/pages/safety.html
@@ -1,7 +1,7 @@
{% extends "layout.html" %}
{% block title %}Safety{% endblock %}
-{% block lead %}Describe all the safety issues of your project.{% endblock %}
+{% block lead %}{% endblock %}
{% block page_content %}
diff --git a/wiki/pages/sponsors.html b/wiki/pages/sponsors.html
index ca47ca32b26e96fa4b723d1f2f96041a40aff5b7..dead5c77a4f4c52eac641405cb30f444e6d4aaef 100644
--- a/wiki/pages/sponsors.html
+++ b/wiki/pages/sponsors.html
@@ -10,7 +10,7 @@
<p>We would like to exalt the immense support of the <b>Biology Institute</b> and <b>Pró-Reitoria de Extensão e Cultura (ProEC) of Unicamp</b>. Without the enormous trust and funding entrusted to our team we would not have had the great opportunity to represent our university internationally, and we hope to return in results at least double of the expectations deposited in us.</p>
- <img src="https://static.igem.wiki/teams/4435/wiki/pages/acknowledgements/logosproecunicamp.png" class="page-img">
+ <img src="https://static.igem.wiki/teams/4435/wiki/pages/acknowledgements/logosproecunicamp.png" class="pages-img">
<p>To the <b>Synthetic Biology Laboratory (LaBS)</b> at Unicamp, we also owe deep thanks for the work structure made available throughout the year during which we developed Cellulopolis.</p>
diff --git a/wiki/pages/team.html b/wiki/pages/team.html
index 44c694f5d47cc1d2d894dc17febf37fc6d742bb0..e6d08bade2633ac66f43c46d8dc5342c3ed7b2a3 100644
--- a/wiki/pages/team.html
+++ b/wiki/pages/team.html
@@ -164,4 +164,12 @@
</div>
</div>
+<hr>
+
+<h1>Team Bonding</h1>
+
+ <p>From the very beginning, the UNICAMP’s iGEM team intended to honor the name of the university where it takes place. The University of Campinas is unique for its diverse and specialized campi, and gathering this diversity of students and ideas. One of our biggest concerns, as the activities were divided between team members , was to make sure all members were always aligned with everything that was happening with the project, even if it wasn’t related to their area. In this way, everyone contributed with their skills while also learning from each other.</p>
+
+ <img class="pages-img" src="https://static.igem.wiki/teams/4435/wiki/team/team-bonding.png">
+
{% endblock %}