Difference between revisions of "Team:Utrecht/"

Line 1,123: Line 1,123:
  
 
<script id="page-safety" type="text/template">
 
<script id="page-safety" type="text/template">
−
<div class="page-heading">Safety</div>
+
<div class="page-heading">OUTCASST Safety information</div>
−
well important init
+
 
 +
Since safety is a very important aspect in synthetic biology, we collaborated with the RIVM National Institute for Public Health and Environment of the Netherlands. They encouraged us to think about safety before we act. We came to the conclusion that safety has different meanings for different stakeholders. Here we describe the most important points for these stakeholders. Besides safety, we’ve also thought about the societal impact of our tool and the possible ethical issues involved. The information we gathered was summarized in an infographic, which we used to talk to the general public about synthetic biology and safety at an event organized by the RIVM.
 +
 
 +
<br><br>
 +
 
 +
<b>INFOGRAPHIC</b>
 +
 
 +
<br><br>
 +
<h2 class="subhead" id="subhead-2">Safety</h2>
 +
 
 +
<b>1.1 Lab Safety</b><br><br>
 +
Since we are working with GMO’s we have to follow several safety regulations to ensure the safety of our team members whilst working on OUTCASST. Our team’s lab management worked closely with drs. Fraukje Bitter-van Asma, the Occupational Health and Safety & Environment Expert of Utrecht University. She helped us determine which safety forms and permits needed to be filled out, filed and requested as well as which university guidelines and emergency measures were in place in case of calamities and to prevent health risks to both team members working on the lab and the environment.
 +
<br><br>
 +
<b>1.2 User Safety</b><br><br>
 +
Since we are going to use OUTCASST for the diagnosis of Chagas disease, our users will be caregivers and medical professionals in rural areas. Safety of our device is of great importance to them. During the interviews to find suitable end users (see the end-users section), we also discussed the safety of HEK293T cells to detect specific DNA regions. All interviewees were unanimous that these cell lines would not be a problem regarding safety issues and that potential risks would lie with the samples applied to the device. Since we are going to use blood from people that are potentially infected with parasites, it is important to point out the risk of contamination of the caregiver or other people. This risk is, however, no different from that of other simple diagnostic procedures.
 +
<br><br>
 +
Since the HEK293T cells are too fragile to use in the device, we opt to use air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki. To make the use of these cells as safe as possible, the design of our tool is going to be a closed system, wherein everything is present and only the blood sample will be applied. There will also be several mechanisms and kill-switches incorporated in the detecting cells. This way, the cells are physically separated from both the user and patients and this minimizes the chance of survival of these cells outside of the system.
 +
<br><br>
 +
<b>1.3 Patient Safety</b><br><br>
 +
With our design, early and rapid diagnosis of Chagas disease is possible and thus effective treatment can take place. Our detection method is relatively non-invasive since only a small blood sample of the patients has to be taken. The design is made out of one piece, which reduces risks during use.
 +
<br><br>
 +
<b>1.4 Public Safety</b><br><br>
 +
An essential part of public safety is providing information about Chagas disease and how our device works. The disease is not contagious but is transmitted by a vector. Currently, all efforts for Chagas prevention are directed at vector control. By limiting the carriers of the disease, infection amongst humans is prevented. Although these strategies do not affect the disease or those infected, it does limit the exposure of uninfected individuals to the pathogen. The OUTCASST tool itself can add to these strategies by helping resolve who are and are not infected with the pathogen, increasing public health and safety further.
 +
<br><br>
 +
Naturally, use of GMO tools needs to be done safely and responsibly, lest it proves a risk to public safety. We have made sure to make our tool robust to handling errors and tried to make it as easy to use as possible. That way, it will be less likely for something to go wrong.
 +
<br><br>
 +
<b>1.5 Environment</b><br><br>
 +
In order to minimize environmental impact of OUTCASST, our closed-box system must be disposed of in a proper manner. Disposing of OUTCASST must adhere to guidelines set for GMO products. These guidelines differ from country to country but since it is best to take clear precautions, we have added a disposal guideline in the toolkit manual.
 +
 
 +
<br><br>
 +
<h2 class="subhead" id="subhead-3">Societal impact</h2>
 +
One of the advantages of our device is that people with an HIV co-infection can now also be tested for infection with Chagas disease. False-negative results, as are common with immune-response detection kits, can be avoided in this and other immunodeficient populations. Another advantage is that the test can be performed immediately when Chagas disease infection is suspected.
 +
<br><br>
 +
With already existing serological tests, detection can only occur when enough antibodies are present in circulation, which takes some time. In some cases, the amount of antibodies produced is too low to be detected with serological tests, which will also give a false-negative result. With our tool, faster diagnosis and hence more effective treatment will contribute to eradication of Chagas disease.
 +
 
 +
<br><br>
 +
<h2 class="subhead" id="subhead-4">Ethical issues</h2>
 +
There are 2 main ethical considerations of usage of the OUTCASST system:
 +
<br>
 +
<ul>
 +
<li />Issues arising from use of GMO
 +
<li />Issues arising from DNA detection
 +
</ul>
 +
 
 +
<br>
 +
 
 +
<b>3.1 Issues arising from use of GMO</b><br><br>
 +
Widespread use of GMO is still not widely accepted by the public. Guarantees of safety, efficiency and overall improvement of public health must be provided to achieve mass appeal. Concerns arise over the possibility of allergenicity, gene transfer and outcrossing. For our sensor, the main issue would be horizontal transfer of our genetic system into foreign cellular bodies, thereby releasing a novel biological machine into the ecosystem. However, there is no obvious advantage gained by the incorporation of a DNA detection mechanism as seen in OUTCASST to native organisms. The expenditure of energy and limited resources to maintain this system would in fact, be a disadvantage. Nevertheless, release of the non-native DNA to the ecosystem would allow the normal evolutionary machinery to access this DNA. Random mutations, insertions and deletions could lead to genotypes which would indeed have new  novel characteristics, which would else not have been present. Therefore the accidental release of the  genetic material  could have unforeseen consequences on the ecosystem and raises ethical questions.
 +
<br><br>
 +
However, the OUTCASST toolkit consists of genetically modified cells stored in a closed box environment. This closed box environment contains the cells in an isolated environment, minimizing the risk of our genetically modified cells escaping into the environment. When disposed of correctly, in line with protocols for disposal of genetically modified material, then there should be no chance of contamination or escape of our gene into the natural environment.
 +
 
 +
<br><br>
 +
 
 +
<b>3.2 Issues arising from DNA detection</b><br><br>
 +
OUTCASST is a diagnostic tool which can be programmed to detect specific sequences of DNA. Thus, OUTCASST is susceptible to issues arising from the ownership of the information within the DNA, and the problems arising from the knowledge of this information. In many cases, the predictive value of the DNA is not fully known, and it may lead to undesired consequences for the patient. Indeed, in cases where no treatment or intervention is available it may be in the best interest of the patient not to screen for a certain gene.
 +
<br><br>
 +
Usage of OUTCASST for detection of pathogen DNA avoids many of the issues arising from the detection of human DNA, notably the issues of information ownership. Because OUTCASST will give a yes / no answer to the presence of pathogenic DNA, it is very much in the interest of all parties to know the answer so as to be able to respond accordingly.
 +
 
 
</script>
 
</script>
  
 
<script id="page-product-design" type="text/template">
 
<script id="page-product-design" type="text/template">
−
<div class="page-heading">Product design</div>
+
<div class="page-heading">Integrated human practices: design of OUTCASST</div>
−
1
+
Altogether, we gathered information from many different fields and perspectives. All of these views help us to optimize our design to use OUTCASST as a tool for diagnosing Chagas disease. Since we want our tool to be easy to use in the field and rural areas, there are different aspects that should be taken into account. Robustness and resistance of the toolkit to temperature fluctuations and humidity are chief among these. In addition, it is important not to rely on material and storage containers such as fridges or freezers (Marit de Wit, Doctors without borders).
 +
<br><br>
 +
 
 +
<b>INTERACTIVE SCREEN</b>
 +
 
 +
<br><br>
 +
 
 +
The tool should be cheap to produce and easy to use. To achieve this goal, the tool needs to have a closed box design wherein only the blood sample has to be added and a simple protocol can be followed to perform the test (Jet Bliek and Ruud van den Bogaard, Academical Medical Center Amsterdam: clinical genetics). Disposal of the system also needs to be considered (collaboration RIVM National Insitute for Public Health and the Environment).
 +
<br><br>
 +
There are also some things in the OUTCASST toolkit that need to be changed in comparison to the experimental approach in order to prepare the system for diagnosing Chagas disease. One of these things is the use of HEK293T cells, which need a very stable environment to stay alive. In the eventual tool, we will need to use cells that are more robust to environment fluctuations yet still cannot survive outside of the device (Patrick van Zon and Pieter-Jaap Krijtenburg, University Medical Center of Utrecht: genome diagnostics.) We also used a fluorescence signal as output in the experiments, which requires a fluorescence microscope to analyse the test results. To avoid the need of these and other equipment, we would ideally use an output signal in the form of visible light or, more promisingly, a change of color that is visible to the naked eye. Another thing we should keep in mind is the time it takes to get the results from our test device (Marit de Wit, Doctors without Borders).
 +
<br><br>
 +
There are also technical aspects that should be considered, like the method used for lysis of the parasites in the sample. Lysis needs to occur to get free DNA, i.e. a hypotonic solution (Jaap van Hellemond, Erasmus University Medical Center Rotterdam: parasitology). If a colorant is used as reporter mechanism, we need to remove the red color of heme groups from red blood cells, too, as it would interfere with the output signal.
 +
<br><br>
 +
Lastly, we should consider the target DNA we want to use to detect the parasites. Things that require careful consideration are GC-content, which has influence on binding affinity and specificity of the guide RNA. Specificity needs to be mutation specific as a strand with different base pairs should, ideally, not activate the system (Hans Bos and Hugo Snippert, University Medical Center Utrecht: cancer research).
 +
<br><br>
 +
The design of OUTCASST is shown below. Here you can click on the different numbers, which will guide you through the use of our tool and the reasons behind each step.
 +
<br><br>
 +
 
 +
<br><br>
 +
<h2 class="subhead" id="subhead-2">Toolkit design solutions</h2>
 +
 
 +
The OUTCASST toolkit has a closed box design, wherein all the components to perform the test are present in distinct compartments, separated by seals. These seals can be broken by applying pressure on them.
 +
<br><br>
 +
As was stated earlier, a lot of variables need to be kept constant to keep the HEK293T cells alive. Because of this, it is not feasible to use these cells in our design. Instead, we opt to use air-dried cells from the anhydrobiotic insect, Polypelidum vanderplanki, which can be stored at room temperature for 251 days and can restart proliferating again after rehydration (Watanabe K, Imanishi S, Akiduki G, Cornette R, Okuda T. Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration. Cryobiology. 2016 Aug 31;73(1):93-8). This way the shelf life of our tool can also be prolonged. To prevent the risk of our GMO getting out in the environment, several mechanisms and kill-switches will be incorporated in the cells, so they can only survive in our closed box system, in their resurgent state. This can be done by manipulating the metabolism, so that the cells can’t produce a crucial substance for survival, which will be added in the toolkit medium. In case the cells get out of the toolkit, they will die because of the absence of the crucial substance.
 +
<br><br>
 +
Rehydration can be done with a suitable medium. This has to be done one hour before use. The seal between the dried insect cells and the medium can be broken to pump the medium manually to the cells. After rehydration, the medium can be manually pumped to the waste compartment.
 +
<br><br>
 +
The next step is to add the two guide RNA’s to the revived cells. The gRNA’s are present in the design as dry powder to prevent premature degradation. This time, two seals need to be broken. First, the gRNA needs to be dissolved with the contents of another medium compartment. Then the medium with gRNA can be pumped to the cells where they will bind to dCas9 and dCpf1 on the extracellular cell membrane. This process takes about 10 minutes and after that, the medium with excessive gRNA can also be pumped to the waste compartment.
 +
<br><br>
 +
These were the preparation steps and the real diagnosis can start now. First off, a blood sample has to be taken from a patient that might be infected with Chagas disease. To prevent the blood from clotting, heparin or EDTA can be added to the sample. The blood sample can then be introduced to the tool, after which the device needs to be sealed. To get access to the parasite DNA, all cells need to be lysed, including the red blood cells. This is done with a lysis buffer, a hypotonic solution.
 +
<br><br>
 +
The next step is to pump everything to a next compartment, wherein there are heme-binding compounds (such as HEBP) linked to the inside surface to decolorize the sample. Then a hypertonic resetting buffer is added to return the sample to isotonic levels, in order to prevent damage to the detector cells.
 +
<br><br>
 +
Now, the seal separating the sample from the cells can be broken and the sample can be introduced to the actual sensor. A color signal will appear after about 10 to 12 hours in case the patiënt is infected and will continue to become more visible after that.
 +
<br><br>
 +
The output signal will be a blue chromoprotein. This way, the sample color will become blue (or purple if there is still a little bit of heme in the sample) upon detection of the targeted DNA sequence.
 +
<br><br>
 +
After use, the tool should be disposed of in a safe manner preventing it to end up in the environment. Therefore, there will be a disposal guideline added in the toolkit manual. The test can be disposed of in a self-sealing bag, which can be boiled after the test is completed to minimize the risks.
 +
 
 +
 
 +
<br><br>
 +
<h2 class="subhead" id="subhead-3">Additional considerations</h2>
 +
 
 +
There are still a lot of things that should be considered to make the OUTCASST tool optimal for diagnosing Chagas disease.
 +
<br><br>
 +
The first thing we still need to consider is the blood sample size needed to perform the test. From the patiënts aspect it would be best to take as little as possible. A smaller blood sample would also mean that the device can be made smaller, which in turn also makes the production costs for one test cheaper. However, there needs to be enough pathogen DNA in the blood sample to make sure that the test gives the good results. It would be possible to pretreat a larger sample to concentrate it before applying, increasing the chance of correct diagnosis, but this would again require skilled professionals and materials.
 +
<br><br>
 +
We have also thought about a question that was raised at the University Medical Center at the Cancer department. The question was why we wanted to express our system on the membrane of eukaryotic cells and not just express it intracellularly in bacteria. Then a blood sample could be added and the bacteria can be heat shocked to get the pathogen DNA intracellular, activating the binding of the two proteins. In this case, there would be a loss of the amplification step, since the transcription factor is then able to activate the reporter gene without a signal or cleavage of the transcription factor. Since we don’t know what the minimum amount of blood needed is, we wanted to design it in the way we can get the most signal, which is to include the amplification step. If it would prove that this amplification step is not needed, we could also just put the proteins in the tool and use a split reporter. On the other hand, the tool would not rely on use of living cells, which would make the use of our tool a whole lot safer
 +
<br><br>
 +
We should also consider the material that the device is going to be made of. It should be of sturdy quality to prevent contamination of the environment with the device’s content. From the production perspective, the costs to produce it should be as low as possible to make the tool affordable. A main issue with costs, currently, is the production of the gRNA as it is expensive to synthesize.
 +
<br><br>
 +
We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. We want to take this information into account to decide the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the T. cruzi parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist).
 +
 
 
</script>
 
</script>
  
Line 1,138: Line 1,247:
  
 
<script id="page-sponsors" type="text/template">
 
<script id="page-sponsors" type="text/template">
−
<div class="page-heading">Sponsors</div>
+
<div class="page-heading">Our sponsors</div>
−
1
+
For our project we have had the luck of generous sponsors who either helped us financially, supplied us with material for our lab-work or helped us with other activities.
 +
<br><br>
 +
<table class="sponsors" width="100%">
 +
<tr>
 +
<td width="300"><img src=""></td>
 +
<td>RIVM is the National Institute for Public Health and the Environment, which belongs to the Ministry of Health, Welfare and Sport of the Dutch government. They offered a sponsorship to the Dutch iGEM teams based on an assignment titled ‘think before you do’, stimulating us to think about the societal implications that our project could have. We submitted a proposal and the RIVM offered us a €1500 grant to further our project.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>DSM is a science-based company focusing on health, nutrition and materials to drive sustainable innovation. DSM is active in many different markets, including medicine, energy and food, so developments in synthetic biology are of major interest to them. For our project, they sponsored us with €1000.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>Snapgene is a software company, providing useful software for molecular biology. Snapgene offers fast and reliable software to visualize and design molecular biology experiments such as cloning and PCR experiments. They sponsored us with full team licenses for the Snapgene software, which we have used extensively during our project.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>eLabjournal is a product by BIO-ITECH BV, a company providing software as a service for research laboratories. Their eLabjournal product offers a simple and effective software environment to keep our lab notes organized and allows easy access  wherever and whenever we need it. eLabjournal sponsored us with full team licenses for their software.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>Takara Bio is the biomedical unit of Takara Holdings Inc. and focuses on revolutionary technologies to improve human health. Takara Bio is composed of three units; the Bioindustry division, the AgriBio division and the Gene Therapy division. Takara Bio Europe has offered us a cloning kit and protein purification kit, both of which were frequently used in our experiments.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>NTrans Technologies is a company based in Utrecht, specializing in delivery of content into cells using the iTOP technology which was developed in the Geijsen lab of the Hubrecht Institute. NTrans offered us guidance and assistance with our project by facilitating a lab technician experienced in the work we had to do for our project. We thank them for Clara Martinez’ amazing assistance and patience with our silly questions.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>Integrated DNA Technologies (IDT) is a company focusing on the development and production of products for life sciences, both for research and diagnostic purposes. They are an important supplier of synthetic DNA & RNA, next generation sequencing services and other tools and products for molecular biology. IDT supports us by offering us 20 kb of free custom DNA.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>MathWorks is the leading company on mathematical computing software. Their two primary products are MATLAB, for the development of algorithms and data-analysis, and Simulink, for designing and running simulations of dynamic, multidomain models. These products are a staple in innovation and development in life sciences as well as other fields. Furthermore, MathWorks’ tools are used in research and educational settings. MathWorks provided us with licenses for their fantastic software and technical support.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>SpeechRepublic offers workshops and programs for the development of communication and presentation skills. There are three elements at the center of their methods: being self-confident, inspiring others and having the courage to bring your message across. They offered our team a free workshop on presenting for four team members. This helps us greatly in preparing for our upcoming presentations, specifically the presentation at the Jamboree in Boston.</td>
 +
</tr>
 +
<tr>
 +
<td><img src=""></td>
 +
<td>New England BioLabs is a company that develops and produces reagents for life sciences research. They offer a wide range of products for molecular biology. They have been generous to many iGEM teams over the years, and supported our team as well this year with a DNA assembly kit.</td>
 +
</tr>
 +
</table>
 
</script>
 
</script>
  
Line 1,352: Line 1,504:
 
"stakeholders" : {},
 
"stakeholders" : {},
 
"safety" : {
 
"safety" : {
−
1: "Safety",
+
1: "OUTCASST Safety information",
−
2: "Hmmmm"
+
2: "Safety",
 +
3: "Societal impact",
 +
4: "Ethical issues",
 
},
 
},
 
"product-design" : {
 
"product-design" : {
−
1: "Product design",
+
1: "Integrated human practices: design of&hellip;",
−
2: "Hmmmm"
+
2: "Toolkit design solutions",
 +
3: "Additional considerations",
 
},
 
},
 
"team" : {
 
"team" : {
Line 1,363: Line 1,518:
 
2: "Hmmmm"
 
2: "Hmmmm"
 
},
 
},
−
"sponsors" : {
+
"sponsors" : {},
−
1: "Sponsors",
+
−
2: "Hmmmm"
+
−
},
+
 
"achievements" : {
 
"achievements" : {
 
1: "Achievements",
 
1: "Achievements",
Line 1,500: Line 1,652:
 
</script>
 
</script>
  
−
<style class="cp-pen-styles">/* quick grid */
+
<style class="cp-pen-styles">
 +
.sponsors td { padding-bottom: 30px; }
 +
 
 +
/* quick grid */
 
.shuffle-container {
 
.shuffle-container {
 
   width: 93%;
 
   width: 93%;

Revision as of 14:29, 16 October 2017

<!DOCTYPE html>

  • Home
  • Experimental
  • Human practices
  • Team & Sponsors


Cas9 & Cpf1 secretion
and activity
Comparison of endonuclease activity for Cas9 and Cpf1 that has been produced in, and excreted by, HEK293 cells.
MESA two-component system replication
Details on the MESA two-component system, explanation of its relation to our design and the results of its reproduction.
OUTCASST system production
Detailed explanation of the OUTCASST mechanism, experimental progress and technical prospects.
Modeling and
mathematics
Ordinary differential equations, cellular automaton and an object based model for optimal linker-length estimation.
InterLab study participation
Results and details of our measurements for the iGEM 2017 InterLab Study.
Stakeholders & opinions
Interviews and dialogues with stakeholders, potential users, third parties and experts relating to pathogen detection or DNA-based diagnostics.
Risks & safety-issues
Implications and design considerations relating to safety in the usage and implementation of OUTCASST as a diagnostics tool.
Design & integration
OUTCASST toolkit and product design with factors such as bio-safety and user-friendliness taken into account.
Meet our team
About us, our interests and roles in the team and our supervisors.
Sponsors
A listing of our sponsors, how they assisted us and our gratitude for their assistance.
Achievements
A short description of all that we have achieved during our participation in the iGEM.
Intro
The problem
The system
The OUTCASST two-component system
This year, Utrecht University participates in the iGEM for the first time. We aim to create a cheap DNA detection kit for disease diagnosis that is easy to use and does not rely on complicated sequencing technologies.

The problem

Disease diagnosis is of great importance for healthcare. In developing countries, diagnoses often have to be made based on limited information, even though accurate disease determination based on pathogen specific DNA sequences is possible through sequencing technologies. These technologies, however, require specialised equipment and expertise that simply is not available everywhere. The OUTCASST two-component system and detection kit hopes to alleviate this problem.
Start
2
3
4

The system

The OUTCASST two-component system consists of two proteins, expressed to the membrane of a dryable cell. One of the proteins is a Cas9-fusion and the other contains Cpf1. Both proteins can be given a guide RNA that makes it bind to a specific, user-chosen, complementary sequence. When both proteins bind a DNA fragment from a sample, they co-localize, so that a transcription factor is released intracellularly which then induces an intracellular reporter mechanism such as a dye or fluorescent signal.
Binding of components with search-specific gRNA sequences.

Next
DNA sample fragment binds to one of the components.

Next
Fragment binding with both components induces co-localization.

Next
Protease cleaves, transcription factor is released from complex.