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  <div class = "container"> <h1 style="text-align:center; font-size: 75px;"><font face= "Poiret One">Results</font></h1><hr>
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<h1 style="text-align:center; font-size: 50px;"><font face= "Poiret One">Isolated LacZ from iGem part BBa_K564012</h2><br>
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            Human practice in iGEM is about reaching out to the general public and enhancing communication with the science community. On this page, we focus on strengthening the exchange of information for a concrete launch of synthetic biology application. In order to accomplish this goal, we have created the Bench-to-Bedside Guide, which provides individuals with recommendations on how to bring a diagnostic test developed in the lab into the real world. To further supplement this general guide, we created one specifically tailored to our diagnostic test, which uses aptamers and NASBA to detect tuberculosis biomarkers. </font></p>
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<p style="text-align:center; font-size:20px;"><font face="verdana">We transformed a vector with LacZ from the registry for further cloning, we need this vector to produce another plasmid with an alpha deletion(M15 mutant).</font><br><hr></p> <img src="https://static.igem.org/mediawiki/2016/7/73/Isolated_LacZ_from_iGem_part_BBa_K564012.png" width="500" height="500" style="float:center">
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            This is an <b> interactive </b> guideline, so please click on the tiles below to expand and learn more! </font></p>
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<h1 style="text-align:center; font-size: 50px;"><font face= "Poiret One">Transferring LacZ into pET28</h2><br>
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<p style="text-align:center; font-size:20px;"><font face="verdana"> In order to have a control for our cell-free expression system, we cloned LacZ into the protein expression plasmid to compare LacZ expression to expression of our system with and without the presence of its target protein. </font><br><hr></p> <img src="https://static.igem.org/mediawiki/2016/e/e8/Transferring_LacZ_into_pET28.png" width="500" height="500" style="float:center">
 
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<h1 style="text-align:center; font-size: 50px;"><font face= "Poiret One">Creation of delta-M15 Mutant</h2><br>
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<p style="text-align:center; font-size:20px;"><font face="verdana"> Successfully deleted the alpha fragment in our Lacz plasmid, leaving only the omega fragment. The omega fragment complements the alpha fragment produced in the proximity-dependent ligation forming functional LacZ. This enzyme can break down X-Gal giving a colorimetric output. The plasmid is a very important part of our system, allowing alpha complementation and therefore producing a color visible to the naked eye. <br><br>
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<a href="https://static.igem.org/mediawiki/2016/f/f8/Sequencing-Results-Using-T7-Primer-alignment_%281%29.pdf" target="_blank">Click to see sequencing results.</a></font><br></p>
 
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<h1 style="text-align:center; font-size: 50px;"><font face= "Poiret One">Proximity-Dependent Ligation Assay in Agarose Gel using SYBR Gold</h2><br>
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            Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis. Around the world, 8 million new tuberculosis cases arise and 2 million lives are taken per year (1). There is not yet an effective point-of-care
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            diagnostic test and current methods of diagnosis tend to be expensive, not sensitive enough and untimely. Additional challenges arise when attempting to diagnose a disease in developing regions, as many current diagnostic tests require highly trained technicians and laboratory equipment, preventing the tests from being done on-site. </font></p>
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Proximity dependent ligation allows our system to only be activated in the presence of its target protein. We decided to test our system using thrombin as a proof of concept.<br><br>
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              <p><font face="Verdana">The market size  of a business needs to be considered when thinking of demographics and segmentation. A common problem is having a market size too large to target for a startup, so a better approach would be to better start at a local level. Segmentation is helpful in narrowing down the focus and the way that the market could be divided. By understanding the demographic background of the entire market, one then uses the relevant information to identify the market to enter first.
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                When diagnosing a widespread disease like tuberculosis, the market is as large as the entire world and includes all age groups because they could all be affected by the disease. Although only 9,412 of over 10 million new cases in 2014 were in the United States (2), tuberculosis is rampant in less developing parts of the world. Over 95% of TB cases and deaths are in developing countries (3). In 2014, the largest number of new TB cases occurred in South-East Asia and Western Pacific regions, accounting for 58% of new cases worldwide. Africa carries the biggest burden, with 281 cases per 100,000 people(3). The six countries that stand out as having the largest number of incident cases in 2014 were India, Indonesia, Nigeria, Pakistan, People’s Republic of China and South Africa (3).
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In this assay we aim to see the probes and connector ligated in the presence of thrombin, this should be about 300 base pairs long. In the absence of thrombin each component would just be floating in the solution unable to bind to each other, the probes are 145 and 163 base pairs long. Since we are trying to see the size of single stranded DNA bands, which can be difficult to visualize with ethidium bromide, we used SYBR Gold dye for staining.  
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We also wanted to test which connector binds the best out of three different length connectors, we set up three reactions without thrombin and three with thrombin, using different connectors, making a total of six reactions. Samples were left incubating at 35C for 9 hours and heated to 70C to deactivate ligase and melt the connector-probe interactions.  
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The first three lanes have the different sizes of connectors in ascending base pair length (going from left to right), no thrombin was added to the first three lines. The next three lanes are identical except they also contain thrombin. The last lane is probe 1 alone without any other reaction components added. The bands observed for probe 1 alone seem to suggest that two common conformations for probe 1, an unfolded conformation which is observed to run at approximately the position of the 100bp band on the ladder, and a folded conformation which runs farther down on the gel. The bands for the ligation reactions do not seem to show significant ligation activity, because there is no clear high molecular weight band; however, there may be a small amount of ligation occurring in lane 5, the reaction with the 3C connector and thrombin added, evidenced by the faint high molecular weight band in that lane.
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            <h3 style="padding: 0px 0px 10px 20px">II. Target Market, Market Need, & Customer Discovery</h3>
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</font><br><hr></p> <img src="https://static.igem.org/mediawiki/2016/8/8c/TestingPLAagarose.JPG" width="500" height="500" style="float:center">
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              <p><font face="Verdana">This section is important as it is where investors are identified!
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                Target market is about identifying and understanding the potential customers, and this is often done by looking at the similar products or services that are already on the market. Market Need serves to highlight the section of the existing products in which specific improvement could be made to satisfy the customers, and this is the key point to be considered for product development and design concept. Sometimes there are no comparable products or services to an invention, then it is necessary to find the potential customers in another way, which is Customer Discovery. </font></p>
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                Current routine methods for diagnosing TB include acid fast staining of clinical material followed by smear microscopy and M. Tuberculosis culture. However, the former has a poor sensitivity (∼70%) and the latter is costly and an untimely procedure (4). Table 1 from Dorman’s paper “New Diagnostic Tests for Tuberculosis: Bench, Bedside, and Beyond” lists some of the more promising new technologies and tests currently in demonstration or late-stage evaluation phase and those endorsed for use by the World Health Organization which are the ones that seem to be the best. However, in general none of these diagnostic tests are perfect, being either untimely, expensive or not sensitive enough. </font></p>
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                The ideal definition of a TB diagnostic test would be one that is a highly accurate, simple to perform, point-of-care test that test urine or blood, and with ability to detect and predict active TB anywhere in the body, in combination with effective preventive and treatment strategies (4). These are all characteristics we sought for Aptatpaper. </font></p>
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                Global Biodiagnostics, an Aptapaper competitor, estimates that the demand for a point-of-care TB diagnostic is of 100-200 million tests a year, since not everyone tested will have the disease (5). Based on this information, we expect our customer pool to be quite large and diverse: primarily hospitals, governments, and nonprofit organizations. On-site testing in pharmacies or grocery stores similar to how vaccines and some blood tests are available at pharmacies in the United States, presents further opportunities for sales. In addition, TB testing is required of all health care workers and volunteers in the United States, which provides another market where the test could be priced higher. </font></p>
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                In 2015, the international community set goals regarding tuberculosis with the endorsement of the Sustainable Development Goals (SDGs) and the World Health Organization’s (WHO) milestones, hoping to culminate in the eradication of TB by 2030 (6). This international endorsement for treatment and diagnosing of TB could increase sales, although at the current rate, this goal would take until 2182 to achieve (6). To eradicate TB, a major shift in how the disease is prevented, diagnosed, and treated will be required. </font></p>
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                Customer discovery continues to be a main focus area for the team. We have been advised by several people, including our iGEM advisor Professor Marcus Ammerlaan,  CEO of Warmilu Grace Hsia, Dr. Krishna Rao and Rama Kannenje, President of the Relief for Africa Foundation. Based on the weakness of competing diagnostic technologies and personal experience, they believe that our tool would be heavily adopted. </font></p>
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                      In an interview with Rama Kannenje, President of the Relief for Africa Foundation, we learned that a cheap and easy diagnostic method is the key to save lives in Kenya. Almost 90% of HIV patients have TB and it is not uncommon for 1 in 5 hospitalized patients to have TB. People in remote areas cannot be diagnosed or treated due to the great distance they would have to travel to get tested and treated. There is also a lack of human resources to do the testing; there are very few doctors so clinical officers (doctor assistants) end up treating thousands of patients alone. Patients have to travel between laboratories and hospitals in order to get tested and treated. Doctors often forego diagnostic tests because patients can't afford them, so they blindly treat for what they suspect is TB. (7) </font></p>
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                      When TB is diagnosed early on, patients receive medication and go on with their lives. Despite government hospitals being generally inadequate, when citizens are diagnosed with TB, the Kenyan government does front the cost for medication which is usually in stock. However, with approximately 60% of the population living on one dollar a day, prevention is not a top priority. If the cost of testing, currently at $100 per person in Kenya, could be reduced and made easily accessible to rural communities, thousands of lives would be saved annually. The missing link here is a rapid diagnostic test that would prevent this cascade of unfortunate events. (7)</font></p>
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                      By observing Kenya’s circumstances, the Michigan Synthetic Biology Team (MSBT) thought there was a window of opportunity here to put synthetic biology to use. The wellbeing of thousands of people around the world could be ensured by improving TB diagnostics through the use of paper-based genetic switches. Thus, Aptapaper was born. It is a complex technology fused into simple strip which is storable at room temperature for up to a year. It produces an easy to read colorimetric output in hours, costs just a few cents per test, requires no additional equipment and minimal training. (7)</font></p>
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              <p><font face="Verdana">In order to make actually launch a product into the market, it is important to consider the potential barriers that could impede this process or the actual success of the product. Such barriers include competition and regulations needed to place the product in the market. </font></p>
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                      Our product falls into  the quickly growing biotech industry. Due to the high cost of research and government approval, it is common for biopharmaceutical companies to go over a decade without generating their own revenue only to be highly profitable later on. In comparison, as our company will be a diagnostic company, not a biopharmaceutical, the costs and timeline will be significantly lower and shorter. Biotech is also unique in that large biotech companies frequently outsource both research and manufacturing to smaller companies dubbed Contract Research Organizations (CROs) and Contract Manufacturing Organizations (CMOs). This trend has given rise to numerous biotech start-ups that only perform research, receiving funding first from federal grants, and then more traditional sources (angel investors, VCs, incubators, etc.) and partnerships with larger biotech companies.</font></p>
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                      Alternatively, biotech companies of varying sizes avoid the infrastructure costs associated with developing manufacturing centers by hiring CMOs. CROs allow companies to reduce the risks they incur by only buying or investing in drug/diagnostic candidates that they think will be successful or even those which have already received FDA approval. SImilarly CMOs allow biotech companies to quickly produce drugs for clinical trials without having to build/expand their own manufacturing site.</font></p>
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                      Kenyan regulation on medical devices in limited. An application process involving testing and certifications is required. Medical equipment in Kenya is almost all imported (8). Since Kenya is in great need of a TB diagnostic test and is used to importing medical devices, the application process would likely be easy.</font></p>
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                      Most medical products have access to Kenya as long as they meet the Kenya Bureau of Standards (KEBS) requirements (8). The Kenyan Government, through KEBS, now requires that all consignments of regulated products entering Kenya must obtain a Certificate of Conformity issued by one of two firms: Société Générale de Surveillance S.A. (SGS), or Intertek. The price of this Certificate can be anywhere between $450 and $2,675 usd (9). Medical device product evaluations can be done by the National Public Health Laboratories and typically involve 400 tests at a cost of about $1000 (9).</font></p>
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              <p><font face="Verdana">Our method is well suited from both humanitarian and business perspectives as it is a common disease, infecting about 10 million new people a year. Since a cure is readily available, governments and nonprofits would be willing to invest in a superior diagnostic tool like ours. The technology would also be easily adapted to diagnose other diseases, massively increasing the potential target market. Our primary customers are hospitals, governments and NGOs, predominantly in sub-Saharan Africa, South-East Asia and Brazil. Aptapaper is affordable, that it is easy to envision on-site TB testing at pharmacies and grocery stores in these countries. The test would be optimal for humanitarian organizations such as Doctors Without Borders, that practice medicine in countries in great need of better healthcare. </font></p>
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<p style="text-align:center; font-size:20px;"><font face="verdana"> We tested the proximity dependent ligation assay using a wider range of conditions, still using an agarose gel and SYBR Gold stain to visualize the DNA bands produced. In the first lane we use a 1kb plus ladder from Thermo Fisher. Lane 2 is a 30 minute reaction at room temperature without thrombin. Lane 3 is a 30 minute reaction at room temperature with thrombin added. Lanes 4 and 5 are the same as lanes 3 and 4 but run at 37 degrees C. Lane 6 is a 4 hour reaction at room temperature without thrombin. Lane 7 is a 4 hour reaction at room temperature with thrombin added. Lanes 8 and 9 are the same as lanes 6 and 7 but run at 37 degrees C. Lane 10 is a 19 hour reaction at room temperature without thrombin. Lane 11 is a 19 hour reaction at room temperature with thrombin added. Lanes 12 and 13 are the same as lanes 10 and 11 but run at 37 degrees C. The results do not seem to show any successful ligation. Interestingly, we see faint red bands of higher molecular weight in the 19 hour reactions which were run at 37 degrees C. </font><br><hr></p>  
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            The next important step is to consider the development of the product in a broad view. This is similar to the scientific method but regarding businesses. There are a set of logical steps to be followed after identifying the target market and doing the market analysis in order to fulfill the process of launching a new product into the market. The steps can be seen in the diagram below.</font></p>
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Latest revision as of 03:41, 20 October 2016

Home Project Modeling Team BioBrick Human Practices Safety Awards

Results

Isolated LacZ from iGem part BBa_K564012


We transformed a vector with LacZ from the registry for further cloning, we need this vector to produce another plasmid with an alpha deletion(M15 mutant).

Transferring LacZ into pET28


In order to have a control for our cell-free expression system, we cloned LacZ into the protein expression plasmid to compare LacZ expression to expression of our system with and without the presence of its target protein.

Creation of delta-M15 Mutant


Successfully deleted the alpha fragment in our Lacz plasmid, leaving only the omega fragment. The omega fragment complements the alpha fragment produced in the proximity-dependent ligation forming functional LacZ. This enzyme can break down X-Gal giving a colorimetric output. The plasmid is a very important part of our system, allowing alpha complementation and therefore producing a color visible to the naked eye.

Click to see sequencing results.

Proximity-Dependent Ligation Assay in Agarose Gel using SYBR Gold


Proximity dependent ligation allows our system to only be activated in the presence of its target protein. We decided to test our system using thrombin as a proof of concept.

In this assay we aim to see the probes and connector ligated in the presence of thrombin, this should be about 300 base pairs long. In the absence of thrombin each component would just be floating in the solution unable to bind to each other, the probes are 145 and 163 base pairs long. Since we are trying to see the size of single stranded DNA bands, which can be difficult to visualize with ethidium bromide, we used SYBR Gold dye for staining.

We also wanted to test which connector binds the best out of three different length connectors, we set up three reactions without thrombin and three with thrombin, using different connectors, making a total of six reactions. Samples were left incubating at 35C for 9 hours and heated to 70C to deactivate ligase and melt the connector-probe interactions. The first three lanes have the different sizes of connectors in ascending base pair length (going from left to right), no thrombin was added to the first three lines. The next three lanes are identical except they also contain thrombin. The last lane is probe 1 alone without any other reaction components added. The bands observed for probe 1 alone seem to suggest that two common conformations for probe 1, an unfolded conformation which is observed to run at approximately the position of the 100bp band on the ladder, and a folded conformation which runs farther down on the gel. The bands for the ligation reactions do not seem to show significant ligation activity, because there is no clear high molecular weight band; however, there may be a small amount of ligation occurring in lane 5, the reaction with the 3C connector and thrombin added, evidenced by the faint high molecular weight band in that lane.

Proximity-Dependent Ligation Assay (extended)


We tested the proximity dependent ligation assay using a wider range of conditions, still using an agarose gel and SYBR Gold stain to visualize the DNA bands produced. In the first lane we use a 1kb plus ladder from Thermo Fisher. Lane 2 is a 30 minute reaction at room temperature without thrombin. Lane 3 is a 30 minute reaction at room temperature with thrombin added. Lanes 4 and 5 are the same as lanes 3 and 4 but run at 37 degrees C. Lane 6 is a 4 hour reaction at room temperature without thrombin. Lane 7 is a 4 hour reaction at room temperature with thrombin added. Lanes 8 and 9 are the same as lanes 6 and 7 but run at 37 degrees C. Lane 10 is a 19 hour reaction at room temperature without thrombin. Lane 11 is a 19 hour reaction at room temperature with thrombin added. Lanes 12 and 13 are the same as lanes 10 and 11 but run at 37 degrees C. The results do not seem to show any successful ligation. Interestingly, we see faint red bands of higher molecular weight in the 19 hour reactions which were run at 37 degrees C.