Difference between revisions of "Team:Warwick/HP/Silver"

Revision as of 18:26, 19 October 2016

iGEM Warwick 2016 - Modelling

Summary

Our sensor is primarily designed to detect bacterial infections caused by members of the spirochete family, including the genera Borrelia and Leptospira that cause Lyme disease and leptospirosis respectively. Our team chose to target these disease due to their widespread nature and high human impact; 330,000 people are estimated to contract Lyme’s disease each year in the US alone[1], with between approximately 7 and 10 million people infected with Leptospirosis annually[2]. As well as these diseases, the versatility of our system allows us to also target detection of heavy metals in water. The metals we chose to focus on were lead and mercury; these metals were selected due to the severity of poisoning symptoms, along with their widespread presence in both developed and developing countries. Our decision to include developing countries in our target market imposed specific restrictions on our product design, as detailed below.

Ethics

At Westminster University’s UK iGEM meet up, Dr Robert Smith from King’s College London gave an enlightening talk concerning the social and ethical dimensions of synthetic biology, and the types of issues the teams should consider as their projects advance. This stimulated a review of our project through an ethical lens, and whether any controversial aspects were justified.

Synthetic biology may provide the solutions to global issues such as lack of clean energy and inefficient drug delivery. However, this innovative technology is accompanied by concerns over the potential for human or environmental harm resulting from genetically engineered organisms. The possible biological threat that could be posed to national security sometimes makes it difficult to determine to what extent research should be censored in order to protect the quality of human or environmental life.

Another common ethical issue associated with synthetic biology is the concept of playing god – is it morally correct that humans should be capable of altering naturally occurring organisms? How appropriate is it for humans to have ownership in the forms of patenting other organisms within a live system? When is it deemed acceptable for living creatures to no longer have rights? When approached from this perspective, it is easy to understand why biological modification of organisms is such a controversial topic, although similar ethical issues also apply to topics such as genetic, stem cell, nanotechnology and neuroscience research.

When discussing the manner in which our detection system should be marketed, we considered whether it was socially correct for people to receive a diagnosis from a piece of paper, rather than another individual. Using the common pregnancy test as an example, we feel there would be little public dispute if our product were to be sold as a self-test. However, following further market research, we decided that it should in fact be stocked in clinics and administered by trained professionals. This would also prevent the issue of bias supply due to pricing.

Having researched the ethical concerns surrounding synthetic biology, we have gained an in depth understanding of the possible disadvantages associated with utilizing this technology. However, in our opinion, by incorporating precautionary steps, for example ‘kill switches’ and other biologically engineered safeguards, the potential for technological innovation and scientific advancement far outweighs the potential risk.

Safety

As our device is a diagnosis tool, it was important that we considered any safety precautions that would be required in order to reach a stage where our device can be tested under real life conditions.

The first safety consideration we made concerned the type of bacteria for which our device would be constructed within. We elected to use E. coli, as it is a risk group 1 organism. Leptospira, the primary bacteria we aim to detect, is a risk group 3 organism banned from use in iGEM. This, in addition to leptospirosis being a contagious disease, led us to compromise our testing procedures to prevent requiring the spirochete itself to be present in the blood sample.

We decided that instead of designing our device to test directly for Leptospira RNA, we would initially design it to test for an analogue RNA that does not code for a specific protein. This avoids having to grow up Leptospira bacteria in our lab, or having to transform another bacteria with a plasmid encoding for Leptospira specific RNA. In this way we eliminate the risk associated with handling infectious bacterial proteins, and prevent the release of a bacteria able to produce Leptospira proteins. Another solution would have been to transform another bacteria with Leptospira specific RNA, but without including an RBS that would enable RNA translation. We decided that if we needed to modify the RNA in this way, it would be simpler and less risky to use an entirely synthetic RNA sequence as the trigger for the device instead.

Another concern we had was that testing the concentration of Leptospira in the blood would require direct contact with either Leptospira bacteria, or human blood. Human blood is a high-risk substance that must be handled with caution. It may contain numerous possible contaminants that may be transmissible to users – especially likely when individuals untrained in the specific safety requirements, as. In training labs in university, students are taught to screen their own blood samples. However, we were hesitant to do this, as in addition to the ethical implications of testing a diagnostic tool on human blood, individual variations in our blood could potentially alter the mechanics of the device. We decided that we would order in pre-screened human blood serum that we personally inoculate with modified bacteria carrying the trigger RNA gene. This avoids having to use both our own unscreened blood, or blood from another source, while also sidestepping the issue of using Leptospira.

The abiotic design of our sensor ensures that there is no risk of releasing modified bacteria into a natural environment. However, release of signalling molecules, such as fluorescent proteins and malachite green, is still possible. Malachite green in particular is known to cause respiratory distress in fish [RN], as well as being moderately cytotoxic[3]. Fluorescent proteins, the alternative to malachite green, are capable of producing reactive oxygen species (ROS) in water upon exposure to ultraviolet light [RN]. In order to keep generation of ROS’s to a minimum, green fluorescent protein (GFP) was selected as a potential signalling molecule, as its confined barrel shape leads to increased quenching of ROS’s compared to other fluorescent proteins [4]. Whilst the use of GFP as our signalling molecule would seem to be more environmentally friendly, it is important to note that a detector based on GFP would require additional incubation at 37 °C for 24 hours whereas malachite green does not. The electricity necessary for this incubation not only reduces the environmental friendliness of the design, but also its suitability for use in a third world setting. It is these additional complications that led our team to incorporate both malachite green and GFP into separate products, based on their expected usage.

Bacterial antibiotic resistance is an ever-persistent concern within the biomedical industry. Although our primary focus is leptospirosis, we realized that the initial design of our system would be far less appropriate for testing for Lyme disease – the original concept for which our product was developed. Lyme disease is treated with a course of tetracycline, but in order for our detection system to be successful it must be resistant to this antibiotic. For this reason, we would have to only allow the product to be utilized by trained professional, or exchange the bacteria for one with a different antibiotic resistance.

Sustainabilty

The overall cost of production and sale of our sensor was an important factor that our team were keen to minimize. We set a final target of 0.50 USD per sensor based on our discussion with David Lloyd, CEO of FREDsense. To achieve this, we decided to use a freeze dried bacterial lysate system, as opposed to a more conventional in solvo test.

Once the team decided to freeze dry our sensor, it became necessary to evaluate the properties of various substrates available for mounting our product, to determine which was most appropriate. The main candidates were paper, nitrocellulose and silica-paper. Each of these substrates had a range of advantages and disadvantages, as detailed in Table 1 below.

Our sensor is primarily designed to detect bacterial infections caused by members of the spirochete family, including the genera Borrelia and Leptospira that cause Lyme disease and leptospirosis respectively. Our team chose to target these disease due to their widespread nature and high human impact; 330,000 people are estimated to contract Lyme’s disease each year in the US alone[1], with between approximately 7 and 10 million people infected with Leptospirosis annually[2]. As well as these diseases, the versatility of our system allows us to also target detection of heavy metals in water. The metals we chose to focus on were lead and mercury; these metals were selected due to the severity of poisoning symptoms, along with their widespread presence in both developed and developing countries.

Table 1: The advantages and disadvantages of utilizing different materials in the hardware of our sensor.

Due to its unstable nature, our team decided that nitrocellulose was unsuitable as a substrate for any real world application. However, both paper and silica-paper showed promise in a wide variety of applications. It was decided that the more expensive, but durable, silica-paper would be used for in tougher field environments and water testing; whilst the cheaper but less robust, ordinary paper would be used for blood tests and domestic uses.

Unfortunately, the design of our sensor does not allow repeat use. It was therefore responsible to review the recyclability and disposal of our product. The non-toxic nature of the cell lysate means that the paper based sensor can be recycled in a conventional manner. However, at present there is no way to recycle the silica-paper based substrate, leading our team to decided that paper would be used in all our products, except when a very robust substrate was required.

The miniaturization of our design not only reduces the amount of raw material needed, but also facilitates the transport of a large number of units at one time. As well as lowering the material and environmental cost, the smaller design allows for easier transport and storage - especially with regards to usage and distribution of field test kits in developing countries.

Intellectual Property Rights

Research advancements within synthetic biology, and the patenting of new systems, could affect the existing trade and global justice systems due to the formation of monopolies. Because of this, rapid development of this technology may only benefit scientists, investors and large businesses, with the advantages to those in developing areas being highly limited due to lack of accessibility. Currently, the UK government dissuades non-competitive business practices, so this problem has less of an impact on our project. As we are using a natural Cas9 sequence and a dCas9 protein, our system does not conflict with any existing patents, such as the initial CRISPR/Cas patent by Feng Zhang in April 2014.

References:
[1] http://www.cdc.gov/lyme/stats/
[2] http://www.who.int/zoonoses/diseases/lerg/en/index2.html
[3]http://apps.webofknowledge.com/full_record.do?product=UA&search_mode=GeneralSearch&qid=34&SID=N163dLMizXq1ZVG7fMr&page=1&doc=1
[4] https://www.ncbi.nlm.nih.gov/pubmed/21359336

@@ Line 68: / Line 68: @@
        <section id="banner">
          <h1>Human Practices Silver</h1>
+      </section>
+    <section id="three" class="wrapper special">
+        <div class="inner">
+          <header class="align-center">
+            <h2>Summary</h2>
+          </header>
+          <div class="flex flex-2">
+            <article class="nopicture">
+              <p>Our sensor is primarily designed to detect bacterial infections caused by members of the spirochete family, including the genera Borrelia and Leptospira that cause Lyme disease and leptospirosis respectively. Our team chose to target these disease due to their widespread nature and high human impact; 330,000 people are estimated to contract Lyme’s disease each year in the US alone[1], with between approximately 7 and 10 million people infected with Leptospirosis annually[2]. As well as these diseases, the versatility of our system allows us to also target detection of heavy metals in water. The metals we chose to focus on were lead and mercury; these metals were selected due to the severity of poisoning symptoms, along with their widespread presence in both developed and developing countries. Our decision to include developing countries in our target market imposed specific restrictions on our product design, as detailed below.</p>
+            </article>
+          </div>
+        </div>
        </section>
-  <!-- Summary -->
      <section id="three" class="wrapper style1 special">
          <div class="inner">
            <header class="align-center">
-             <h2>Summary</h2>
+             <h2>Ethics</h2>
+          </header>
+          <div class="flex flex-2">
+            <article class="nopicture">
+              <p>At Westminster University’s UK iGEM meet up, Dr Robert Smith from King’s College London gave an enlightening talk concerning the social and ethical dimensions of synthetic biology, and the types of issues the teams should consider as their projects advance. This stimulated a review of our project through an ethical lens, and whether any controversial aspects were justified.<br /><br />Synthetic biology may provide the solutions to global issues such as lack of clean energy and inefficient drug delivery. However, this innovative technology is accompanied by concerns over the potential for human or environmental harm resulting from genetically engineered organisms. The possible biological threat that could be posed to national security sometimes makes it difficult to determine to what extent research should be censored in order to protect the quality of human or environmental life.<br /><br />Another common ethical issue associated with synthetic biology is the concept of playing god – is it morally correct that humans should be capable of altering naturally occurring organisms? How appropriate is it for humans to have ownership in the forms of patenting other organisms within a live system? When is it deemed acceptable for living creatures to no longer have rights? When approached from this perspective, it is easy to understand why biological modification of organisms is such a controversial topic, although similar ethical issues also apply to topics such as genetic, stem cell, nanotechnology and neuroscience research.<br /><br />When discussing the manner in which our detection system should be marketed, we considered whether it was socially correct for people to receive a diagnosis from a piece of paper, rather than another individual. Using the common pregnancy test as an example, we feel there would be little public dispute if our product were to be sold as a self-test. However, following further market research, we decided that it should in fact be stocked in clinics and administered by trained professionals. This would also prevent the issue of bias supply due to pricing.<br /><br />Having researched the ethical concerns surrounding synthetic biology, we have gained an in depth understanding of the possible disadvantages associated with utilizing this technology. However, in our opinion, by incorporating precautionary steps, for example ‘kill switches’ and other biologically engineered safeguards, the potential for technological innovation and scientific advancement far outweighs the potential risk.</p>
+            </article>
+          </div>
+        </div>
+      </section>
+    <section id="three" class="wrapper special">
+        <div class="inner">
+          <header class="align-center">
+            <h2>Safety</h2>
+          </header>
+          <div class="flex flex-2">
+            <article class="nopicture">
+              <p>As our device is a diagnosis tool, it was important that we considered any safety precautions that would be required in order to reach a stage where our device can be tested under real life conditions.<br /><br />The first safety consideration we made concerned the type of bacteria for which our device would be constructed within. We elected to use E. coli, as it is a risk group 1 organism. Leptospira, the primary bacteria we aim to detect, is a risk group 3 organism banned from use in iGEM. This, in addition to leptospirosis being a contagious disease, led us to compromise our testing procedures to prevent requiring the spirochete itself to be present in the blood sample.<br /><br />We decided that instead of designing our device to test directly for Leptospira RNA, we would initially design it to test for an analogue RNA that does not code for a specific protein. This avoids having to grow up Leptospira bacteria in our lab, or having to transform another bacteria with a plasmid encoding for Leptospira specific RNA. In this way we eliminate the risk associated with handling infectious bacterial proteins, and prevent the release of a bacteria able to produce Leptospira proteins. Another solution would have been to transform another bacteria with Leptospira specific RNA, but without including an RBS that would enable RNA translation. We decided that if we needed to modify the RNA in this way, it would be simpler and less risky to use an entirely synthetic RNA sequence as the trigger for the device instead.<br /><br />Another concern we had was that testing the concentration of Leptospira in the blood would require direct contact with either Leptospira bacteria, or human blood. Human blood is a high-risk substance that must be handled with caution. It may contain numerous possible contaminants that may be transmissible to users – especially likely when individuals untrained in the specific safety requirements, as. In training labs in university, students are taught to screen their own blood samples. However, we were hesitant to do this, as in addition to the ethical implications of testing a diagnostic tool on human blood, individual variations in our blood could potentially alter the mechanics of the device. We decided that we would order in pre-screened human blood serum that we personally inoculate with modified bacteria carrying the trigger RNA gene. This avoids having to use both our own unscreened blood, or blood from another source, while also sidestepping the issue of using Leptospira.<br /><br />The abiotic design of our sensor ensures that there is no risk of releasing modified bacteria into a natural environment. However, release of signalling molecules, such as fluorescent proteins and malachite green, is still possible. Malachite green in particular is known to cause respiratory distress in fish [RN], as well as being moderately cytotoxic[3]. Fluorescent proteins, the alternative to malachite green, are capable of producing reactive oxygen species (ROS) in water upon exposure to ultraviolet light [RN]. In order to keep generation of ROS’s to a minimum, green fluorescent protein (GFP) was selected as a potential signalling molecule, as its confined barrel shape leads to increased quenching of ROS’s compared to other fluorescent proteins [4].  Whilst the use of GFP as our signalling molecule would seem to be more environmentally friendly, it is important to note that a detector based on GFP would require additional incubation at 37 °C for 24 hours whereas malachite green does not. The electricity necessary for this incubation not only reduces the environmental friendliness of the design, but also its suitability for use in a third world setting. It is these additional complications that led our team to incorporate both malachite green and GFP into separate products, based on their expected usage.<br /><br />Bacterial antibiotic resistance is an ever-persistent concern within the biomedical industry. Although our primary focus is leptospirosis, we realized that the initial design of our system would be far less appropriate for testing for Lyme disease – the original concept for which our product was developed. Lyme disease is treated with a course of tetracycline, but in order for our detection system to be successful it must be resistant to this antibiotic. For this reason, we would have to only allow the product to be utilized by trained professional, or exchange the bacteria for one with a different antibiotic resistance.
+</p>
+            </article>
+          </div>
+        </div>
+      </section>
+       <section id="three" class="wrapper style1 special">
+        <div class="inner">
+          <header class="align-center">
+            <h2>Sustainabilty</h2>
            </header>
            <div class="flex flex-2">
              <article class="nopicture">
-               <p>1) Established a general idea of how our system works (Metal Aptamer sensor and Leptospirosis detector).<br/><br/>2) Constructed useful reaction schemes to represent how our system works.<br/><br/>3) Derived differential equations from reaction scheme using mathematical laws.<br/><br/>4) Estimated reaction constants by reading through related literature and using acquired information to make reasonable estimations.<br/><br/>5) Implemented differential equations with related reaction constants into MATLAB as functions.<br/><br/>6) Produced graphical representations showing fluctuations in molecule number of each species.<br/><br/>7) Devised potential experiments that could be conducted to characterise/parameterise model more accurately.</p>
+               <p>The overall cost of production and sale of our sensor was an important factor that our team were keen to minimize. We set a final target of 0.50 USD per sensor based on our discussion with David Lloyd, CEO of FREDsense. To achieve this, we decided to use a freeze dried bacterial lysate system, as opposed to a more conventional in solvo test.<br /><br />Once the team decided to freeze dry our sensor, it became necessary to evaluate the properties of various substrates available for mounting our product, to determine which was most appropriate. The main candidates were paper, nitrocellulose and silica-paper. Each of these substrates had a range of advantages and disadvantages, as detailed in Table 1 below.<br /><br />Our sensor is primarily designed to detect bacterial infections caused by members of the spirochete family, including the genera Borrelia and Leptospira that cause Lyme disease and leptospirosis respectively. Our team chose to target these disease due to their widespread nature and high human impact; 330,000 people are estimated to contract Lyme’s disease each year in the US alone[1], with between approximately 7 and 10 million people infected with Leptospirosis annually[2]. As well as these diseases, the versatility of our system allows us to also target detection of heavy metals in water. The metals we chose to focus on were lead and mercury; these metals were selected due to the severity of poisoning symptoms, along with their widespread presence in both developed and developing countries.</p>
+              <img src="https://static.igem.org/mediawiki/2016/3/3a/T--Warwick--SilverTable.png">
+              <p>Table 1: The advantages and disadvantages of utilizing different materials in the hardware of our sensor.<br /><br />Due to its unstable nature, our team decided that nitrocellulose was unsuitable as a substrate for any real world application. However, both paper and silica-paper showed promise in a wide variety of applications. It was decided that the more expensive, but durable, silica-paper would be used for in tougher field environments and water testing; whilst the cheaper but less robust, ordinary paper would be used for blood tests and domestic uses.<br /><br />Unfortunately, the design of our sensor does not allow repeat use. It was therefore responsible to review the recyclability and disposal of our product. The non-toxic nature of the cell lysate means that the paper based sensor can be recycled in a conventional manner. However, at present there is no way to recycle the silica-paper based substrate, leading our team to decided that paper would be used in all our products, except when a very robust substrate was required.<br /><br />The miniaturization of our design not only reduces the amount of raw material needed, but also facilitates the transport of a large number of units at one time. As well as lowering the material and environmental cost, the smaller design allows for easier transport and storage - especially with regards to usage and distribution of field test kits in developing countries.</p>
              </article>
            </div>
@@ Line 86: / Line 129: @@
-  <!-- Introduction -->
      <section id="three" class="wrapper special">
          <div class="inner">
            <header class="align-center">
-             <h2>Introduction</h2>
+             <h2>Intellectual Property Rights</h2>
            </header>
            <div class="flex flex-2">
              <article class="nopicture">
-               <p>The Warwick iGEM team decided to use a CRISPR/Cas9 system to build a sensing device for Leptospirosis detection. CRISPR technology is a relatively new, not fully understood technology, so for our project to be successful, it was imperative that we have as detailed an understanding of the way the technology operates as possible. As a result, we spent a lot of time trying to develop a model that accurately represents the behaviour of our system. </p>
+               <p>Research advancements within synthetic biology, and the patenting of new systems, could affect the existing trade and global justice systems due to the formation of monopolies. Because of this, rapid development of this technology may only benefit scientists, investors and large businesses, with the advantages to those in developing areas being highly limited due to lack of accessibility. Currently, the UK government dissuades non-competitive business practices, so this problem has less of an impact on our project. As we are using a natural Cas9 sequence and a dCas9 protein, our system does not conflict with any existing patents, such as the initial CRISPR/Cas patent by Feng Zhang in April 2014.<br /><br />References:<br />[1] http://www.cdc.gov/lyme/stats/<br />[2] http://www.who.int/zoonoses/diseases/lerg/en/index2.html<br />[3]http://apps.webofknowledge.com/full_record.do?product=UA&search_mode=GeneralSearch&qid=34&SID=N163dLMizXq1ZVG7fMr&page=1&doc=1<br />[4] https://www.ncbi.nlm.nih.gov/pubmed/21359336
-              <h3>How the system works/What parts we decided to model?</h3>
+</p>
-              <p>For us to be able to model our system, we had to establish what we thought would be happening while our device was in action. Before our system could start sensing Leptospirosis RNA, we had to produce the three plasmids that in turn produced the sensing complex. Once these plasmids had been constructed, the sensing process could begin.<br/><br/>At the start of the sensing process, when our system is in any environment, Plasmid 1 produces the dCas9. Plasmid 1 is also capable of making GFP. Plasmid 2 produces the RNA binding protein with the attached Effector while Plasmid 3 makes the sgRNA. All of these products are released into the surrounding environment in large quantities.<br/><br/>Our sgRNA design incorporates a region that is complementary to dCas9 molecules that we call the dCas9 handle. When the sgRNA is in its original shape, the handle is not accessible to the dCAs9, however, if there is Leptospirosis RNA present in the environment, it will bind to the sgRNA, changing its shape and allowing the dCAs9 to bind to it.<br/><br/>The dCas9/sgRNA complex is guided back to Plasmid 1 by the dCas9 component. When the dCas9/sgRNA complex is bound to Plasmid 1, the dCas9 molecule then acts as a beacon for the RNA binding protein with the attached effector to find the sgRNA and bind to it.<br/><br/>When the previous binding process is complete, the RNA binding protein/effector recruits an RNA polymerase, allowing it to read the gene for GFP production present on Plasmid 1. GFP is produced and is released into the surrounding environment.<br/><br/>When establishing the way that our system operates, we made the following assumptions to make the modelling process easier:<br/>1) Each step of the process happens one step at the time (so two species  binding simultaneously does not happen).<br/>2) The RNA being sensed has a strong binding affinity to our sgRNA and will not unbind once bound.<br/>3) The sgRNA will be produced in relative abundance relative to the other species in the environment.<br/><br/>After having established the way we expected our system to perform, we decided that it would be most useful to model the rate of change of concentration for the different molecules that would be present in our system at any given time.<br/><br/> Our ultimate goal was to accurately predict the amount of GFP being produced. This would give us an insight into how sensitive our detection device will be with differing amounts of Leptospirosis RNA present in a system. </p>
              </article>
            </div>