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| − | <h1 class="text-center">Project Design</h1> | + | <div class="col-sm-8"> |
| − | <h2>Introduction</h2> | + | <h2 class="text-center">Introduction</h2> |
| − | <h5>It was important to us that our design was as uncomplicated and elegant as possible. To effectively diagnose Chagas disease, we needed a system that could be performed in a field setting. This eventually pushed us in the direction of a cell-free system, inspired partly by the work of Keith Pardee in diagnosing the Zika virus cell-free.<br/> | + | <h5>Chagas disease is a neglected tropical disease named after a Brazilian scientist, Carlos Chagas, who first described the life cycle of the parasite – Trypanosoma cruzi (T.cruzi) that causes the disease. Chagas is primarily transmitted via the faeces of triatomine bugs, when they take a blood meal. Other forms of transmission include:blood transfusions orally via ingestion of contaminated fluids vertical transmission Chagas is endemic to Latin America but increased migration of infected people has led to it spreading to non-endemic countries, consequently increasing the number of people susceptible to the disease and causing it to be a growing global concern. </h5> |
| | + | <h5>Since the 1990s, strategies to reduce the impact of Chagas in endemic countries have largely focused on preventing transmission through vector control programmes and blood banks. Although these achievements have significantly reduced its incidence, they are not sufficient to combat the spread of the disease vertically from a mother to her child. Therefore, congenital Chagas Disease is growing in epidemiological importance, as it is now one of the most persistent form of the transmission among the human population, with prevalence in some rural areas of Bolivia being as high as 70.5%. <br /> <br /><br /> </h5> |
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| − | <br/>Synthetic biology presents many possible paths, and we chose to investigate two of them: one DNA-based, one protein-based. The DNA-based system uses the TetR repressor, and the protein-based system uses OmpA and SpyTag/SpyCatcher to tether proteins to the outer membrane. | + | <img class="img-responsive" style="margin-left: -50px" src="https://static.igem.org/mediawiki/2017/4/49/T--oxford--chagas_disease--life_cycle.png"> |
| − | </h5>
| + | <h3 class="text-center">Parasitology</h3> |
| − | <h2>Background</h2>
| + | <h6>T.cruzi follows the life-cycle shown in the diagram above: </h6> |
| − | <h5>There are many issues associated with developing a diagnostic for a neglected tropical disease. The method has to be (1) effective, (2) affordable, and (3) viable to perform and store within existing infrastructure. Additionally it needs to be accepted by the local community as a reliable and safe method for them to use. Currently, diagnostics for congenital Chagas disease do not sufficiently meet these requirements.
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| − | <br/><br/>Synthetic biology is a currently underutilised as an option for diagnostics. Many tests use enzyme linked immunoassays (ELISAs), which can be expensive and difficult to conduct in some environments. They also rely on having common molecular epitopes, which may not be present for some diseases. Synthetic biology allows us to detect in a different way, and also circumvent the use of antibodies in some cases. It can also lead to smaller, faster, and cheaper detection. We conducted a survey of the general public and found that a majority were in favour of the development of a diagnostic using synthetic biology.
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| − | </h5>
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| − | <h2>Cruzipain as a biomarker</h2>
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| − | <h5>Most current tests for Chagas disease focus on detecting the whole parasite, T. cruzi, its DNA, or antibodies produced in response to its biomarkers. We looked at a traditionally difficult biomarker, cruzipain(ref), a specific cysteine protease. Most tests failed to use it because it has many isoforms and no stable epitope, however its substrate was always the same, and we decided this was ideal to use for our system. This is because it is secreted by the parasite, and as the parasite is present in relatively high concentrations during the acute phase of the disease, there are likely to be relatively high concentrations of cruzipain present. It is also highly specific to T. cruzi meaning that we are unlikely to get false positives from this aspect. By looking at the protease directly we also circumvent a traditional problem which is diagnosis of newborn babies, who do not have fully developed immune systems and can therefore not use immunological tests. This developed our focus on congenital diagnosis. We developed two systems to detect it - one is DNA-based, one is OMV-based.</h5>
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| − | <h2>Design-Build-Test cycle</h2>
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| − | <h5>We used the engineering cycle from Imperial 2011 to help with the design, build, and testing of our system. This cycle shows how interactions with modellers, consumers, and the public contribute to the development of a system, and integrate with the wet lab work. The most important part is it is cyclical in nature, and a design may go through many cycles before it is finished. This constant dialogue is key to creating a product that matches the needs of the consumer as well as being scientifically sound. It allows for iterative improvement of the device.</h5>
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| − | <h2>First DBT cycle</h2>
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| − | <h5>Originally we envisioned producing hirudin directly from our system. However, results from our modelling data (see here) suggested that this would not be fast enough to prevent blood clotting. Thus, we needed an amplification system to increase the speed of hirudin production, and we settled on doing this by releasing it from a sterically hindered system by cleavage by TEV protease. From there, we considered the options of how we could control the levels of TEV protease in the system.</h5>
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| − | <h2>DNA approach</h2>
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| − | <h5>To detect cruzipain, its proteolytic function should lead to a detectable output.
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| − | This leads to two logical options - a positive output and a negative output. As the system is DNA-based, and we want to control the production of an mRNA that would then be translated, this suggests either using an activating or repressing transcription factor. Cleavage by the protease to allow an activator to work would be a positive output, and cleavage to disable a repressor would be a negative output. After research, we concluded that it would be more feasible to produce a negative output using proteolytic cleavage.</h5>
| + | <h5>All life stages of T.cruzi secrete a specific protease, known as cruzipain, which allow the presence of the trypomastigotes to be detected by our biosensor. However, the levels of trypomastigotes in the human blood falls with time after infection, as shown in figure 2. </h5> |
| − | <h5>Therefore we decided to research repressor proteins which could potentially be inactivated by cleavage from cruzipain. After reading several options, we decided on the TetR repressor(ref) as it was shown to be a very strong repressor, and had an easily accessible linker region between its two domains which we could modify to have a cruzipain recognition sequence.</h5>
| + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/2/2c/T--oxford--chagas_disease--evolution-chart.png"> |
| − | </div>
| + | <h6>Figure 2. Scheme of evolution of T.cruzi trypomastigotes in the blood of a human host </h6> |
| − | </div>
| + | <h5>Within 4-8 weeks of being bitten, adults move from the acute phase of Chagas disease to the chronic phase if untreated. </h5> |
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| + | <h5>During the chronic phase, cruzipain levels are very low in the blood and cost effective diagnosis of adults focuses on detecting antibodies specific to T.cruzi. However, antibody based diagnosis is unsuitable for newborns who lack a fully developed immune system. Newborns infected with congenital Chagas disease remain in the acute phase for up to 9 months, during which period there is no current cost-effective diagnostic available. We hope to fill this gap in the ability to diagnose congenital Chagas disease in newborns, using synthetic biology to create a specific protease detection system. </h5> |
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/ e/ e3/T--oxford-- design-- TetR.png" ></img> | + | |
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/b/b9/T--oxford--design--tetrkey.png"></img>
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| − | <h5>TetR acts as a dimer(ref), and each monomer has a DNA binding domain and a dimerisation domain(ref)., consequently the Tet promoter has two TetR binding sites, both needed for full repression of transcription(ref). We took the DNA sequence of TetR Class B from BBa_K106669 and located the linker region using PyMOL and PDB entry 3zqi. The linker region was long enough to modify into a protease recognition sequence.</h5>
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| − | <h5>Due to the dangers of working with a protein from a pathogen, we decided to use a common laboratory protease instead to model our system and provide a proof-of-concept. Therefore, the TetR part we have designed (C200) has a Tobacco Etch Virus (TEV) protease recognition site instead of a cruzipain recognition site. The TEV protease is well-characterised(ref) and can be expressed in simple bacterial systems, making it ideal for testing our sensor.
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/7/73/T--oxford--design--TEVkey.png"></img>
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| − | <h5>We also believe that this version of TetR provides more versatility for relieving repression of a gene or other DNA sequence, as an alternative to adding tetracycline.
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| − | <br/><br/>As a control, we also created a part with an unmodified TetR repressor (C250), which would give no output when the TEV protease is added. This enables us to understand the background rate of transcription and consequently the rate of false positive diagnoses from the repressor being ‘leaky’. Both TetR parts were created with CFP as fusion proteins so we could visualise them in a fluorescence plate-reader. | + | <h3>Symptoms and current diagnosis</h3> |
| | + | <h5>Diagnosis of Chagas disease is difficult, as the disease is mostly asymptomatic in the acute phase and for the majority of the chronic phase. However, prolong onset of the chronic phase leads to 30% of patients develop cardiac disorders and up to 10% develop digestive, neurological or mixed alterations that cause 1200 deaths per year. |
| | + | The main diagnostic methods currently used to diagnose Chagas are summarised in the table below:</h5> |
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| − | <br/><br/>In addition to the TetR parts, we also created auxiliary parts to test them. We have designed a non-self-cleavable TEV protease from BBa_K1319004 with a His tag and an mCherry, and a DNA part which contains a medium-strength tet operator from BBa_R0040 in front of an RBS and eYFP (from AddGene).
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| − | <br/><br/>The combination of CFP, mCherry, and YFP was chosen because they have very limited overlap in their absorption and emission frequencies, meaning it was easier to confidently quantify them relative to one another. </h5>
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| − | <h3>Overall, our proof-of-concept system consists of three parts:
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| − | </h3>
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| − | <ul>
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| − | <li>C100 - TEV protease, with or without mCherry, expressed in pBAD33</li>
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| − | <li>C200 - TetR with a TEV protease cleavage site, with or without CFP, expressed in pQE-60<br/>(C250 - TetR without a TEV protease cleavage site, with or without CFP, expressed in pQE-60)</li>
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| − | <li>C300 - Tet promoter, followed by an RBS and the sequence for YFP, expressed in pQE-60</li></ul>
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| − | <h2>Experimental Plan to test designs</h2>
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| | </div> | | </div> |
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| | + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/7/76/T--oxford--chagas_disease--chagas_disease.jpeg"> |
| | + | <br/><br/> |
| | + | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/7/7f/T--oxford--chagas_disease--stage_of_chagas.png"> |
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| − | <h6 class="text-center">We add IPTG to the cells to induce expression in pQE-60 of the TetR repressor, which then binds to the Tet site upstream of YFP.</h6>
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/e/e2/T--oxford--design--ExpCF2.png"></img>
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| − | <h6 class="text-center">We remove IPTG and add arabinose to induce expression from pBAD33. This then produces a small amount of TEV protease in the system.</h6>
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| − | <h6 class="text-center">TEV protease then cleaves TetR at the linker.</h6>
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/c/c5/T--oxford--design--ExpCF4.png"></img>
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| − | <h6 class="text-center">TetR falls off the promoter region for the gene encoding YFP, allowing RNA polymerase to bind, leading to the production of YFP.</h6>
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| − | <h6 class="text-center">Lots of YFP is produced, and the small amount of TEV protease is amplified into a large signal. The fluorescence can be detected and quantified.</h6>
| + | |
| − | <h3>Experiments and data:</h3>
| + | |
| − | <ul>
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| − | <li>C300 in expression vector to look at efficiency of the promoter
| + | |
| − | <ul>
| + | |
| − | <li>Compare with C300 in shipping vector</li>
| + | |
| − | </ul>
| + | |
| − | </li>
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| − | <li>Measure YFP in C300 to find the time to detectable levels and its max expression</li>
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| − | <li>Use C250 to see how much TetR we need for repression of C300
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| − | <ul>
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| − | <li>Vary expression of C250 to find minimum TetR needed</li>
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| − | <li>Only requires pQE-60 plasmid with C250 and C300</li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li>Measure YFP and CFP with varied levels of inducer and time course in the plasmid with C200/250 and C300
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| − | <ul>
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| − | <li>Compare C250 and C200 to check C200 is still functional</li>
| + | |
| − | </ul>
| + | |
| − | </li>
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| − | <li>Produce and purify TEV</li>
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| − | <li>Experiments with two plasmids
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| − | <ul>
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| − | <li>With optimal amount of C200/C250 induced, vary levels of C100 expressed</li>
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| − | <li>Measure YFP, CFP and mCherry and compare YFP between C200 and C250</li>
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| − | <li>Find levels of C100 that work to determine sensitivity in cells</li>
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| − | <li>Vary time course to find optimal time for the system</li>
| + | |
| − | </ul>
| + | |
| − | </li>
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| − | <li>Compare above experiment to one with tetracycline instead of C100 vector to see how effective TEV protease relief of repression is</li>
| + | |
| − | <li> Purify TetR</li>
| + | |
| − | <li>Compare YFP levels for C200 and C250 in the above experiments with C200 and C250 with their fluorophores removed in the same experiments</li>
| + | |
| − | </ul>
| + | |
| − | <h3 class="text-center">Advantages and Disadvantages of the Cell-Free DNA System</h3>
| + | |
| − | <table class="table table-hover"> | + | |
| | <thead> | | <thead> |
| | <tr> | | <tr> |
| − | <th>Advantage</th>
| + | <th>Test</th> |
| − | <th>Disadvantage</th>
| + | <th>How it works</th> |
| − | </tr>
| + | <th>Benefits</th> |
| − | </thead>
| + | <th>Limitations</th> |
| − | <tbody>
| + | <th>Suitable for newborns</th> |
| − | <tr>
| + | </tr> |
| − | <td>Amplification</td>
| + | </thead> |
| − | <td>Requires a large amount of cell machinery</td>
| + | <tbody> |
| − | </tr>
| + | <tr> |
| − | <tr>
| + | <td>Whole parasite microscopy</td> |
| − | <td>No cells involved</td>
| + | <td> |
| − | <td>Chance of DNA damage</td>
| + | <ul> |
| − | </tr>
| + | <li>Preparation of Giesma blood smears</li> |
| − | <tr>
| + | <li>Visualised using light microscopy</li> |
| − | <td>Cheap</td>
| + | </ul> |
| − | <td>Chance of plasmid uptake by pathogens</td>
| + | </td> |
| − | </tr>
| + | <td> |
| − | </tbody>
| + | <ul> |
| − | </table>
| + | <li>Established method</li> |
| − | <h2>OMV System</h2>
| + | <li>Carried out by already trained professionals</li> |
| − | <h5>Having identified some of the problems associated with the cell-free DNA- based system, we innovated a system that overcame these.
| + | </ul> |
| | + | </td> |
| | + | <td> |
| | + | <ul> |
| | + | <li>Not suitable for the when there is little T.cruzi in the blood</li> |
| | + | <li>Not always possible to differentiate between T.cruzi from T.rangeli, which does not cause disease in humans.</li> |
| | + | </ul> |
| | + | </td> |
| | + | <td>Yes</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td>Polymerase Chain Reaction (PCR)</td> |
| | + | <td> |
| | + | <ul> |
| | + | <li>Molecular detection of T. cruzi DNA is performed using a combination of three real-time PCR assays.</li> |
| | + | <li>Acceptable specimen types are EDTA blood, heart biopsy tissue or cerebrospinal fluid.</li> |
| | + | </ul> |
| | + | </td> |
| | + | <td> |
| | + | <ul> |
| | + | <li>Allows high sensitivity in the acute phase</li> |
| | + | <li>Allows the presence of T.cruzi to be accurately distinguished from T.rangeli</li> |
| | + | <li>Allows direct detection of infection and easy interpretation of results</li> |
| | + | </ul> |
| | + | </td> |
| | + | <td> |
| | + | <ul> |
| | + | <li>High variation in accuracy and lack of international quality controls</li> |
| | + | <li>High cost and complexity means it is not practical to use in a clinical practice</li> |
| | + | <li>Further validation is needed to prove whether PCR is suitable to diagnose the chronic phase of Chagas</li> |
| | + | </ul> |
| | + | </td> |
| | + | <td>Yes</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td>Serological tests</td> |
| | + | <td> |
| | + | <ul> |
| | + | <li>Detection of antibodies against T.cruzi</li> |
| | + | <li>Includes techniques such as indirect fluorescent antibody (IFA) test, a commercial enzyme immunoassay (EIA) and immunochromatographic tests</li> |
| | + | </ul> |
| | + | </td> |
| | + | <td> |
| | + | <ul> |
| | + | <li>Can be used for acute phase and chronic phase</li> |
| | + | <li>High specificity and sensitivity</li> |
| | + | <li>Commercialised and approved for use by WHO</li> |
| | + | <li>Low-cost formats are available</li> |
| | + | </ul> |
| | + | </td> |
| | + | <td> |
| | + | <ul> |
| | + | <li>Cross reactivity can occur with diseases, such as leishmaniasis and schistosomiasis </li> |
| | + | <li>Performance of these tests is lower than reported by their manufacturer, especially against specific strains of T. cruzi</li> |
| | + | <li>Not suitable for immunocompromised patients and newborns</li> |
| | + | </ul> |
| | + | </td> |
| | + | <td>No</td> |
| | + | </tr> |
| | + | </tbody> |
| | + | </table> |
| | + | <h5>The table highlights the lack of a rapid and feasible diagnostic for congenital Chagas disease. Moreover, in June 2016 the WHO and experts on Chagas disease based in Latin America regarded a point of care diagnostic for congenital Chagas as their top priority in terms of the diagnostic needs for Chagas disease. The diagnostic needs were ranked following considerations of existing diagnostic tools and the expected clinical and epidemiological scenario of Chagas disease in the next five years. </h5> |
| | + | <h3>Treatment</h3> |
| | + | <h5>An 8 week course of benznidazole or nifurtimox can be used to kill the parasite and treat Chagas disease. The younger the patient and the closer to acquisition of the infection, the higher the probability of parasitologic cure. Therefore, newborns with congenital Chagas disease have the greatest chance for cure, with data from Argentina indicating that the cure rate is higher than 90% if treatment is given within the first year of life. In most cases the potential benefits of medication in curing, preventing or delaying the Chagas is balanced against the possible adverse reactions that occurring in up to 40% of treated patients. However, newborns are least affected by side effects of benznidazole or nifurtimox, due to the lower weight-accounted dosage, making treatment a very viable option. If the chronic phase is left untreated, additional specific treatment for cardiac or digestive manifestations may be required.</h5> |
| | + | <h4>References</h4> |
| | + | <h6>http://www.who.int/chagas/strategy/en/ <br/> https://www.hindawi.com/journals/bmri/2014/401864/ <br/> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618875/ <br/> |
| | + | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552304/ <br/> https://www.ncbi.nlm.nih.gov/pubmed?cmd=Search&doptcmdl=Citation&defaultField=Title%20Word&term=Marin-Neto%5Bauthor%5D%20AND%20Pathogenesis%20of%20chronic%20Chagas%20heart%20disease |
| | + | <br/> http://emedicine.medscape.com/article/214581-treatment?pa=8NzeMcIsf2L2NYDeJvy4ZoO1LWEPOwX7vVdtvcVUJoOYwMyg61ZQ3PAOKQ1pIwNHbOMFnZcMllAKcR9rY0RREHf7Bj2Gvk6BKC47oRZ1BB8%3D </h6> |
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| − | <br/><br/>In our DNA-based system, TEV protease is activated in the presence of cruzipain; this mechanism relied, however, on DNA expression. We decided to generate an entirely protein-based circuit that acts in a similar way.
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| − | <br/><br/>We initially considered sterically inhibiting TEV protease in the absence of cruzipain. However, due to the crystal structure of the protease, specifically the location of the N- and C-termini far from the active site, we decided this would not be a reliable method, with respect to false positives. We then discovered split TEV protease (Wehr et al.), in which two haves of the protein are present individually in an unconnected, and therefore inactive form. The halves are attached to dimerisation partners (with cruzipain/TEV protease cleavage sites in the linkers, in our device) to prevent spontaneous activation. In the presence of cruzipain, a protein circuit activating TEV protease molecules is induced: the TEV protease fragments are able to dimerize and amplify the signal. We combined these ideas with ones presented in a paper using split proteins in a protease detector (Shekhawat et al.) to generate the design, shown below. We generated two TEV protease fragments, each of which are bound to leucine zipper coils (A, B, and B*). | + | </div> |
| − | </h5>
| + | </div> |
| − | <h5>It is more entropically favorable for the B* coil to bind to the A coil, due to its position, and therefore dimerisation of the A and B coils is inhibited. Once the linker (A–B*) is cleaved, A binds to B, the enthalpically favored reaction. Two of the Leu residues in the B* leucine zipper have been mutated to Ala residues, decreasing the enthalpic benefit of binding to A (another Leu zipper) by approximately 0.2kJ/mol.</h5>
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| − | <h5>In this system, both our input (cruzipain) and our intermediate output (TEV protease) are proteases. We introduced an amplification step to improve the final output (active hirudin) of our circuit – the produced TEV protease will cleave A–B* linkers, thus increasing levels of TEV protease and inactive hirudin, to activate it. As a result, our system will require only a small initial signal to produce a clear response.</h5>
| + | |
| − | <h5>Due to the protein-based nature of our circuit, we would likely not be able to replicate it in a functional cell-free extract. We found a solution in outer membrane vesicles (OMVs). OMVs are lipid vesicles (made from the outer membrane) containing periplasmic solution and proteins constitutively made by all Gram-negative bacteria. A recent paper by Alves et al. demonstrated that enzymatic function could be protected during freeze-drying cycles by containing the enzyme in an OMV. We aimed to target the proteins in our circuit to the bacterial outer membrane, extract OMVs containing them, freeze-dry the OMVs for storage and transport, and finally re-solubilise and lyse the OMVs for diagnosis.
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| − | | + | |
| − | <br/><br/>As the bacterial outer membrane transport system is still not yet fully understood, we adopted the technique described in Alves et al.: (1) OmpA, a membrane protein in E. coli which is known to be transported into OMVs at a high rate, is fused to SpyTag and (2) the functional circuitry component is fused to SpyCatcher and a TorA leader sequence. The TorA leader sequence will transport our protein into the periplasmic space (via the Tat translocase system), where SpyTag and SpyCatcher can form an isopeptide bond and fuse the two parts (1 and 2). The circuit proteins are then taken up into the OMV with OmpA. This system is shown in the diagram below.
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| − | </h5>
| + | |
| − | <h5>Once in the OMVs, they can be freeze-dried, and then resolubilised when the diagnostic is needed. As our test was a blood test this would happen as soon as the blood was added. The OMVs then need to be lysed to expose the protein system within. Our proposal for this is to add powdered detergent to the protein system so that when it is solubilised the detergent can lyse the outer membrane vesicles. In order to test OMV lysis without lysing protein we designed the two parts shown below:</h5>
| + | |
| − | <h5>As we realised that speediness of the diagnostic was important, we began looking for systems specifically designed for a quick response. We were inspired by nature, where short term regulation is almost always mediated solely through protein-protein interactions. While this lost some of the flexibility of the modular and easily programmable DNA system, we knew that our biomarker was a protease, and so provided some programmability in the sequence of the cleavage site. In its proteolytic activity there was already a significant change in protein structure, and so we sought to design a system that could be activated by cleavage.
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| − | As this was a protein based system, we recognised that freeze-drying the system from a cell free extract in the way we did with the DNA based system would be unlikely to be possible. So we looked for alternative ways we could make the protein system cell free, and settled on outer membrane vesicles (OMVs). Outer membrane vesicles are lipid vesicles shed by all gram-negative bacteria. They also take up many outer membrane proteins. We identified this as a potential chassis for our system as a recent paper described the protection of enzymatic activity when an enzyme had been taken up into an OMV and then freeze dried <put paper title here>. Our idea for a cell free method of expressing a protein-based system would be to target our proteins to a bacterial outer membrane, extract the OMVs, freeze-dry the OMVs for storage and transport of our device, and finally to resolubilize and lyse the OMVs when the protein system is needed.
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| − | <br/><br/>We knew that our output needed to generate activate TEV protease to be able to cleave an inactive form of hirudin to an active form. However, as this system could not use transcription or translation, the TEV protease had to be already present in an inactive form. At first we looked to see if we could sterically hinder the TEV protease molecule, which could be relieved by cleavage from the cruzipain enzyme. However, looking at the structure shown below we decided this would not be reliable enough.
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| − | <h1>Design</h1>
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| − | Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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| − | This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:Oxford/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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| − | <li>Explanation of the engineering principles your team used in your design</li>
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| − | <li>Discussion of the design iterations your team went through</li>
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| − | <li>Experimental plan to test your designs</li>
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| − | <h5>Inspiration</h5>
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| − | <li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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| − | <li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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| − | <li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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