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| <a href="https://2016.igem.org/Team:Warwick/Description">Project</a> | | <a href="https://2016.igem.org/Team:Warwick/Description">Project</a> |
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− | <a href="https://2016.igem.org/Team:Warwick/Project">Overview</a> | + | <a href="https://2016.igem.org/Team:Warwick/Description">Description</a> |
| <a href="https://2016.igem.org/Team:Warwick/Design">Design</a> | | <a href="https://2016.igem.org/Team:Warwick/Design">Design</a> |
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| <a href="https://2016.igem.org/Team:Warwick/HP/Gold">Gold</a> | | <a href="https://2016.igem.org/Team:Warwick/HP/Gold">Gold</a> |
| <a href="https://2016.igem.org/Team:Warwick/Integrated_Practices">Integrated Practices</a> | | <a href="https://2016.igem.org/Team:Warwick/Integrated_Practices">Integrated Practices</a> |
− | <a href="https://2016.igem.org/Team:Warwick/Engagement">Education</a> | + | <a href="https://2016.igem.org/Team:Warwick/Education">Education</a> |
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| <h1>Design</h1> | | <h1>Design</h1> |
| <p>CRISPR/Cas9</p> | | <p>CRISPR/Cas9</p> |
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− | <h2>The CRISPR-Cas9 system: Step by step DNA cutting in nature </h2> | + | <h2>The CRISPR-Cas9 System: Step by Step DNA Cutting in Nature </h2> |
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| <h3>Genome Editing: A step by step CRISPR guide</h3> | | <h3>Genome Editing: A step by step CRISPR guide</h3> |
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| <li>Introducing sections of DNA with similar ends to both sides of the cut allows homology-directed repair to occur, allowing the insertion of the introduced DNA into the cut site.</li> | | <li>Introducing sections of DNA with similar ends to both sides of the cut allows homology-directed repair to occur, allowing the insertion of the introduced DNA into the cut site.</li> |
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− | <h2>Advantages and Disadvantages of CRISPR technologies in gene editing</h2> | + | <h2>Advantages and Disadvantages of CRISPR Technologies in Gene Editing</h2> |
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| <p>CRISPR technology is one of the most recently developed gene editing tools. Its predecessors include ZNFs (Zinc Finger Proteins) and TALENs (Transcription Activator-like Effector Nucleases). The advantages of CRISPR technology compared to these previous methods include:</p> | | <p>CRISPR technology is one of the most recently developed gene editing tools. Its predecessors include ZNFs (Zinc Finger Proteins) and TALENs (Transcription Activator-like Effector Nucleases). The advantages of CRISPR technology compared to these previous methods include:</p> |
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| <p>However, as with many existing methods, there are some drawbacks to this form of gene editing. If the sgRNA is not fully complementary to the DNA sequence, it may be unable to bind, preventing the required transcription of the gene. There is also an issue with off-target binding, however as we are using the modified nuclease-deficient Cas9 the potential harm caused by off-target binding is greatly reduced. In systems where highly specific binding is an absolute necessity, as it currently exists, CRIPSR/Cas9 may have too frequent off-target binding for it to be reliable enough in these situations. However there are multiple developments in CRISPR technology to reduce the level of off-target binding: reducing the length of the crRNA can lower off-target binding by reducing overall binding efficiency; or using two single-strand cutting Cas9 targeting opposite strands at the same site to create the double strand cut so that two systems need to fail before an off-target cut occurs.</p> | | <p>However, as with many existing methods, there are some drawbacks to this form of gene editing. If the sgRNA is not fully complementary to the DNA sequence, it may be unable to bind, preventing the required transcription of the gene. There is also an issue with off-target binding, however as we are using the modified nuclease-deficient Cas9 the potential harm caused by off-target binding is greatly reduced. In systems where highly specific binding is an absolute necessity, as it currently exists, CRIPSR/Cas9 may have too frequent off-target binding for it to be reliable enough in these situations. However there are multiple developments in CRISPR technology to reduce the level of off-target binding: reducing the length of the crRNA can lower off-target binding by reducing overall binding efficiency; or using two single-strand cutting Cas9 targeting opposite strands at the same site to create the double strand cut so that two systems need to fail before an off-target cut occurs.</p> |
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− | <h2>Use of CRISPR/Cas9 in our system</h2> | + | <h2>Use of CRISPR/Cas9 in Our System</h2> |
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| <p>The Warwick iGEM 2016 team dedicated a lot of time to researching novel technologies that were potential project focal points. We elected to use CRISPR technology as it possesses a myriad of potential uses, whilst being a very precise method of genetic manipulation. Given that this field of research is relatively new, we were excited by the concept of investigating potential novel applications of the CRISPR/Cas9 system.</p> | | <p>The Warwick iGEM 2016 team dedicated a lot of time to researching novel technologies that were potential project focal points. We elected to use CRISPR technology as it possesses a myriad of potential uses, whilst being a very precise method of genetic manipulation. Given that this field of research is relatively new, we were excited by the concept of investigating potential novel applications of the CRISPR/Cas9 system.</p> |
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− | <h2>Selecting An Activation Method/Activation of genes using CRISPR/Cas9</h2> | + | <h2>Selecting An Activation Method/Activation of Genes Using CRISPR/Cas9</h2> |
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| <p>Our system of gene activation consists of 3 parts that interact and guide activation to specific regions of the E. coli genome. The activation is done by effector proteins that are guided to the gene that reports the activation. In order to guide the effector protein it is fused to a RNA binding protein (RNABP) that recognises and binds a specific RNA motif. This RNA motif is present in our extended sgRNA molecule that interacts with the CRISPR/Cas9 system targeting upstream of the promoter of the gene to be activated. The sgRNA, and by extension the RNABP-effector fusion, is guided to the target gene by a nuclease-deficient Cas9 enzyme (dCas9). The reporter gene we are using is GFP, that when activated produces a green fluorescent protein that is measurable as a colour change in the cell.<br/><br/>We are testing 3 different effector proteins, and 3 different RNA binding domains in order to maximise activation by comparing activity between 9 different effector-RNABP fusions. </p> | | <p>Our system of gene activation consists of 3 parts that interact and guide activation to specific regions of the E. coli genome. The activation is done by effector proteins that are guided to the gene that reports the activation. In order to guide the effector protein it is fused to a RNA binding protein (RNABP) that recognises and binds a specific RNA motif. This RNA motif is present in our extended sgRNA molecule that interacts with the CRISPR/Cas9 system targeting upstream of the promoter of the gene to be activated. The sgRNA, and by extension the RNABP-effector fusion, is guided to the target gene by a nuclease-deficient Cas9 enzyme (dCas9). The reporter gene we are using is GFP, that when activated produces a green fluorescent protein that is measurable as a colour change in the cell.<br/><br/>We are testing 3 different effector proteins, and 3 different RNA binding domains in order to maximise activation by comparing activity between 9 different effector-RNABP fusions. </p> |
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− | <h2>Choosing parts</h2> | + | <h2>Choosing Parts</h2> |
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| <p>Our system requires the collaboration of many proteins that are not naturally found together. It was therefore important that these proteins were selected very carefully to give our project the greatest chance of success. In the design phase, we considered many candidate parts and combinations. Through a selection process dependent upon the fulfilment of target criteria, and the results of rigorous experimentation, we decided upon our final design. The criteria used to choose the proteins were their use in previous working systems, their tropism in nature, and the strength of the RNA binding.<br/><br/></p> | | <p>Our system requires the collaboration of many proteins that are not naturally found together. It was therefore important that these proteins were selected very carefully to give our project the greatest chance of success. In the design phase, we considered many candidate parts and combinations. Through a selection process dependent upon the fulfilment of target criteria, and the results of rigorous experimentation, we decided upon our final design. The criteria used to choose the proteins were their use in previous working systems, their tropism in nature, and the strength of the RNA binding.<br/><br/></p> |
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| <h3>RNA Binding Proteins</h3><p>The RNA binding protein (RBP) recognises and binds to a specific RNA sequence. Our CRISPR system will incorporate these RNA sequences in the gRNA, enabling the RBP to specifically target the site where the dCas9 has bound. The RNA binding proteins that we ultimately selected for experimentation were taken from the Zalatan paper, where they were fused with chromatin modifying proteins and targeted towards the 3' end of the gRNA. </p> | | <h3>RNA Binding Proteins</h3><p>The RNA binding protein (RBP) recognises and binds to a specific RNA sequence. Our CRISPR system will incorporate these RNA sequences in the gRNA, enabling the RBP to specifically target the site where the dCas9 has bound. The RNA binding proteins that we ultimately selected for experimentation were taken from the Zalatan paper, where they were fused with chromatin modifying proteins and targeted towards the 3' end of the gRNA. </p> |
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| <p>A nucleotide linker was added between the RNA binding motif and the Cas9 handle-terminator structure in order to introduce physical space between the binding sites of the dCas9 enzyme and the RNA binding protein. This was introduced to prevent interference with the binding of either protein by the presence of the other.</p> | | <p>A nucleotide linker was added between the RNA binding motif and the Cas9 handle-terminator structure in order to introduce physical space between the binding sites of the dCas9 enzyme and the RNA binding protein. This was introduced to prevent interference with the binding of either protein by the presence of the other.</p> |
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− | <p>A nucleotide linker was added between the RNA binding motif and the Cas9 handle-terminator structure in order to introduce physical space between the binding sites of the dCas9 enzyme and the RNA binding protein. This was introduced to prevent interference with the binding of either protein by the presence of the other.</p> | + | <p>In order to make the system activate only upon the detection of specific foreign RNA (dubbed sRNA), the structure of either the Cas9 handle, crRNA region, or the RNA binding motif must be disrupted when no sRNA is present. Then in the presence of sRNA the structure must then reform.<br/><br/>Based on our previous issues with maintaining the structure of the Cas9 handle, it as decided that the structure of the RNA binding motif would be disrupted. This was done by introducing a stretch of RNA between the first terminator loop and the binding motif that would bind to the nucleotides making up the motif disrupting the structure. Further nucleotides were introduced at either end of this disrupting sequence that were complimentary to the sRNA that was to be detected. When the sRNA is introduced to the system, these complimentary regions (dubbed sensing regions) would bind to the sRNA, preventing the disrupting region from binding to the motif, allowing the motif to reform its original structure.<br/><br/> This was modelled on Nupack in order to determine the lengths of the disrupting region and the two sensing regions. The binding energy of the disrupting region must be greater than the binding energy ====of the motif, and the binding energy of both sensing regions must be greater than that of the disrupting region.</p> |
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− | <h2>Modelling detection of sRNA</h2> | + | <h2>Activation via Aptamers</h2> |
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| <div class="flex flex-2"> | | <div class="flex flex-2"> |
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− | <p>In order to make the system activate only upon the detection of specific foreign RNA (dubbed sRNA), the structure of either the Cas9 handle, crRNA region, or the RNA binding motif must be disrupted when no sRNA is present. Then in the presence of sRNA the structure must then reform.<br/><br/>Based on our previous issues with maintaining the structure of the Cas9 handle, it as decided that the structure of the RNA binding motif would be disrupted. This was done by introducing a stretch of RNA between the first terminator loop and the binding motif that would bind to the nucleotides making up the motif disrupting the structure. Further nucleotides were introduced at either end of this disrupting sequence that were complimentary to the sRNA that was to be detected. When the sRNA is introduced to the system, these complimentary regions (dubbed sensing regions) would bind to the sRNA, preventing the disrupting region from binding to the motif, allowing the motif to reform its original structure.<br/><br/> This was modelled on Nupack in order to determine the lengths of the disrupting region and the two sensing regions. The binding energy of the disrupting region must be greater than the binding energy ====of the motif, and the binding energy of both sensing regions must be greater than that of the disrupting region.</p> | + | <p>The previous system works only for detecting sRNA. In order to detect metal ions, incorporating a metal ion aptamer that disrupts the structure of the sgRNA under normal circumstances and reforms the structure when a metal ion binds was necessary. However it was unfeasible to use any RNA folding prediction program to predict the folding of the metal aptamer when the metal has bound. From papers it was clear that the ordinarily linear aptamer scrunches up into a complex that was impossible to predict. From this it was decided that attempting to reform either the Cas9 handle or the RNA binding protein motif was unlikely. But, forming a structure with the aptamer and the crRNA region similarly prevents sgRNA activity and 3' additions to the sgRNA do not affect Cas9 binding. By adding the aptamer to the 3' end of the sgRNA it can bind to the crRNA preventing activity, and as it is only within the proximity of a single structure it is less likely to cause structural issues when ions bind. When an ion binds the aptamer fold in on itself, unbinding the crRNA.</p> |
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− | <h2>Activation by Aptamers</h2> | + | <h2>Metal Aptamers</h2> |
| </header> | | </header> |
| <div class="flex flex-2"> | | <div class="flex flex-2"> |
| <article> | | <article> |
− | <p>The previous system works only for detecting sRNA. In order to detect metal ions, incorporating a metal ion aptamer that disrupts the structure of the sgRNA under normal circumstances and reforms the structure when a metal ion binds was necessary. However it was unfeasible to use any RNA folding prediction program to predict the folding of the metal aptamer when the metal has bound. From papers it was clear that the ordinarily linear aptamer scrunches up into a complex that was impossible to predict. From this it was decided that attempting to reform either the Cas9 handle or the RNA binding protein motif was unlikely. But, forming a structure with the aptamer and the crRNA region similarly prevents sgRNA activity and 3' additions to the sgRNA do not affect Cas9 binding. By adding the aptamer to the 3' end of the sgRNA it can bind to the crRNA preventing activity, and as it is only within the proximity of a single structure it is less likely to cause structural issues when ions bind. When an ion binds the aptamer fold in on itself, unbinding the crRNA.</p> | + | <p>Initially, our group elected to construct detection systems for the free ion forms of mercury, arsenic and lead, due to the significant impact they have on human life and other ecological systems. Despite the lack of affordable sensors at present, we found that the aptamers for binding these elements were well documented and readily available, so we chose to incorporate these into our design.<br/><br/> In order to produce a metal detecting biosensor, it is necessary to modify our existing sgRNA to make it compatible with metal aptamers. In a recent paper it was found [4] that appending nucleotides to the 5’ end of the sgRNA did not significantly affect the binding of Cas9. In our design, these additional nucleotides are complementary to the targeting region of the sgRNA, so forming an additional stem and loop that represses dCas9 activity. By incorporating known metal binding aptamers into the new sensing region formed by the extra nucleotides, the presence of specific metal ions results in a conformational change. This results in the de-repression of our system, allowing dCas9 to bind and the reporter genes to be activated.<br/><br/> The left aptamer binds mercury via the complexation of the C4-carbonyl of two uracils and the divalent mercury(II) cation. When mercury is not present, the aptamer is a linear unfolded RNA strand. However, up to ten mercury ions can be bound by each aptamer, with the RNA folding into a symmetrical ‘mercury locked hairpin’[4]. At a pH greater than 7.5, hydroxide ions interfere with the likelihood of a metal ion binding with its aptamer. This is because a hydroxide complex forms, reducing the effective mercury concentration. A similar concept applies in a pH less than 7 – the nitrogen atoms within uracil become protonated, reducing its affinity with Hg2+. For this reason, our detector would be most sensitive within the pH range of 7 and 7.5.<br/><br/> The aptamer on the right binds lead (II) ions through ionic interaction with eight guanines, to form a lead-guanine quadruplex. Like the mercury aptamer, the lead aptamer is naturally unfolded, but binds a single Pb2+ ion in a square prismatic structure using the guanine C6 carbonyls. Other metals, such as potassium and sodium, are also capable of triggering quadruplex formation. However, they have a much lower affinity and form less stable structures. Addition of cyanide, thiocyanate or 18-crown-6-ether significantly increase aptamer selectivity for lead.<br/><br/>[3] [http://www.who.int/mediacentre/factsheets/fs379/en/ (World Health Organisation accessed on 30/07/2016) https://www.dropbox.com/home/iGEM%202016/Metal%20ap.%20project?preview=MERCURY_DATA_PAPER.pdf[4] L.Trasande, P. J. Landrigan, C. Schechter, Environ. Health Perspect., 2005, 113, 590 – 596</p> |
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| <p>When developing a frontline diagnosis tool, the timing of diagnosis is critical when considering options for treatment. Current Lyme disease, and other spirochete disease, testing techniques require samples to be sent to an analytical lab – delaying results, potentially leading to complications for the patient. Less economically developed places in the world do not have access to these specialist laboratories, preventing the poor who are more likely to come into contact with tick-borne diseases from being diagnosed and subsequently treated. Furthermore analytical tests are expensive to carry out, separating populations from treatment based on their wealth.<br/><br/> To counteract this, we designed a portable and stable framework which allows the test to be conducted anywhere in the world, cheaply and quickly. This would enable the test to be more widely and immediately available to a greater demographic. Paper based sensing has been tested on diagnosis kits for Zika virus and Ebola, it is only natural that the technology becomes cheaper over time so that more and more diseases will be diagnosable by these tests. While currently freeze-drying an in vitro transcription/translation kits carries a considerable set up cost, the equipment required is already common in well-stocked labs. Cost is further reduced by the small size of the sensors, increasing the throughput of each cycle of freeze-drying reducing the cost per test. Being able to mass-produce diagnosis tools is far preferable to lengthy individual diagnoses that currently dominate. <br/><br/> The paper based sensor in our design consists of a freeze-dried transcription/translation system that contains the genes encoding our device and the compounds that allow it to report activation, while being able to be stored en masse in dry areas at room temperature. This makes transport and storage of testing kits cheap and easy, allowing people in areas that may lack refrigeration to keep a stock of tests at all times. Samples can be collected and tested at the same time, so that transporting samples to labs is unnecessary and diagnosis is immediate. </p> | | <p>When developing a frontline diagnosis tool, the timing of diagnosis is critical when considering options for treatment. Current Lyme disease, and other spirochete disease, testing techniques require samples to be sent to an analytical lab – delaying results, potentially leading to complications for the patient. Less economically developed places in the world do not have access to these specialist laboratories, preventing the poor who are more likely to come into contact with tick-borne diseases from being diagnosed and subsequently treated. Furthermore analytical tests are expensive to carry out, separating populations from treatment based on their wealth.<br/><br/> To counteract this, we designed a portable and stable framework which allows the test to be conducted anywhere in the world, cheaply and quickly. This would enable the test to be more widely and immediately available to a greater demographic. Paper based sensing has been tested on diagnosis kits for Zika virus and Ebola, it is only natural that the technology becomes cheaper over time so that more and more diseases will be diagnosable by these tests. While currently freeze-drying an in vitro transcription/translation kits carries a considerable set up cost, the equipment required is already common in well-stocked labs. Cost is further reduced by the small size of the sensors, increasing the throughput of each cycle of freeze-drying reducing the cost per test. Being able to mass-produce diagnosis tools is far preferable to lengthy individual diagnoses that currently dominate. <br/><br/> The paper based sensor in our design consists of a freeze-dried transcription/translation system that contains the genes encoding our device and the compounds that allow it to report activation, while being able to be stored en masse in dry areas at room temperature. This makes transport and storage of testing kits cheap and easy, allowing people in areas that may lack refrigeration to keep a stock of tests at all times. Samples can be collected and tested at the same time, so that transporting samples to labs is unnecessary and diagnosis is immediate. </p> |
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− | <h2>Metal Aptamers</h2>
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− | </header>
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− | <div class="flex flex-2">
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− | <article>
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− | <p>Initially, our group elected to construct detection systems for the free ion forms of mercury, arsenic and lead, due to the significant impact they have on human life and other ecological systems. Despite the lack of affordable sensors at present, we found that the aptamers for binding these elements were well documented and readily available, so we chose to incorporate these into our design.<br/><br/> In order to produce a metal detecting biosensor, it is necessary to modify our existing sgRNA to make it compatible with metal aptamers. In a recent paper it was found [4] that appending nucleotides to the 5’ end of the sgRNA did not significantly affect the binding of Cas9. In our design, these additional nucleotides are complementary to the targeting region of the sgRNA, so forming an additional stem and loop that represses dCas9 activity. By incorporating known metal binding aptamers into the new sensing region formed by the extra nucleotides, the presence of specific metal ions results in a conformational change. This results in the de-repression of our system, allowing dCas9 to bind and the reporter genes to be activated.<br/><br/> The left aptamer binds mercury via the complexation of the C4-carbonyl of two uracils and the divalent mercury(II) cation. When mercury is not present, the aptamer is a linear unfolded RNA strand. However, up to ten mercury ions can be bound by each aptamer, with the RNA folding into a symmetrical ‘mercury locked hairpin’[4]. At a pH greater than 7.5, hydroxide ions interfere with the likelihood of a metal ion binding with its aptamer. This is
| + | |
− | because a hydroxide complex forms, reducing the effective mercury concentration. A similar concept applies in a pH less than 7 – the nitrogen atoms within uracil become protonated, reducing its affinity with Hg2+. For this reason, our detector would be most sensitive within the pH range of 7 and 7.5.<br/><br/> The aptamer on the right binds lead (II) ions through ionic interaction with eight guanines, to form a lead-guanine quadruplex. Like the mercury aptamer, the lead aptamer is naturally unfolded, but binds a single Pb2+ ion in a square prismatic structure using the guanine C6 carbonyls. Other metals, such as potassium and sodium, are also capable of triggering quadruplex formation. However, they have a much lower affinity and form less stable structures. Addition of cyanide, thiocyanate or 18-crown-6-ether significantly increase aptamer selectivity for lead.<br/><br/>[3] [http://www.who.int/mediacentre/factsheets/fs379/en/ (World Health Organisation accessed on 30/07/2016) https://www.dropbox.com/home/iGEM%202016/Metal%20ap.%20project?preview=MERCURY_DATA_PAPER.pdf[4] L.Trasande, P. J. Landrigan, C. Schechter, Environ. Health Perspect., 2005, 113, 590 – 596</p>
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| <div class="copyright"> | | <div class="copyright"> |
− | © Warwick iGem 2016. | + | © Warwick iGEM 2016. |
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− | <a href="https://2016.igem.org/Team:Warwick" class="logo" > | + | <p>We thankfully acknowledge generous funding support from our sponsors below.</p> |
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