Difference between revisions of "Team:Newcastle/InterLab"

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                             <h3 class="subhead">Newcastle InterLab Study</h3>
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                 <h1 class="display-1">OVERVIEW</h1>
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                 <h1 class="display-2">2018 Interlab study aims</h1>
 
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                    <p style="font-size:100%">Reproducibility, the ability to carry out and replicate the results of a single experiment, is an important aspect of scientific disciplines. However, in the life sciences, the concept of reproducibility has become a large problem. A vast number of experiments throughout the various disciplines of the life sciences are seen to lack a reproducible nature, ultimately costing and inadvertently wasting large sums of money. Synthetic biology is no exception to the troubles of reproducibility, with inaccurate part characterisation impacting the the ability to use Bio-Design Automation (BDA) to build fully functioning, novel synthetic gene circuits.</p>
 
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                    <p style="font-size:100%">iGEM devised the inter-lab study – an annual, large-scale study carried out by institutions around the world by researchers of varying experience levels – to determine the reproducibility of individual synthetic biology protocols, assessing and addressing the limiting factors of reproducibility. The study attempted to address variation between different models of plate readers by producing a step by step protocol for measurement and analysis, allowing the production of directly comparable fluorescence measurements. In addition, ribosome binding site (RBS) sequences designed to increase precision of expression were included in the devices to be transformed into the host cells.</p>
 
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                    <p style="font-size:100%">A weakness in the measurement of fluorescence relative to OD600, as with previous IGEM interlab protocols, is the potential discrepancy between optical density and actual cell concentration. This year the IGEM study aims to reduce lab-to-lab variability further by measuring GFP fluorescence relative to absolute cell counts or colony forming units. Normalisation of fluorescence to colony forming units goes further by allowing measurement of fluorescence relative only to viable cells, and thus a more accurate measurement of promoter strength, whereas OD600 and absolute cell count measures cannot differentiate between viable and non-viable cells.</p>
 
 
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                <p class="about-para">Once the project idea was finalised, the team began looking for cheap, efficient and standardised methods for growing plants in iGEM. The hope was that such an item existed that would meet these specifications as well as being a closed container to prevent contamination and also providing a high throughput of plants. It was soon established that such an item did not exist to meet our specifications. Therefore, to combat this issue, it was decided that the best way forward would be to design our own hydroponics system. This would allow us to grow large amounts of Arabidopsis in a controlled setting for the purposes of our project. Several team members were assigned to this “sub-project”.</p>
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                <p class="about-para">Before getting hands-on in building the system, the team as a whole established a few design parameters. For example, the system needed to be cheap and easy to build from scratch. This is so future iGEM teams are able to construct the system for their own needs and even build upon our design, as necessary. Additionally, the system must be versatile, open-source and easily adapted for various conditions such as light intensity and wavelength. By adopting such an open and adaptable design the intention is that the end-user is able to effortlessly match the system to their needs, without getting entangled in streams of code.</p>
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                <p class="about-para">Several weeks were spent modifying the design until a design was found that met all the above criteria, the specifications of the design can be seen below.</p>
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                <h5>UP TO</h5>
 
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                <div class="stats__count">1344</div>
 
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                <h5>SEEDS CAN BE GROWN</h5>
 
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                <h5>IN HYDROPONICS</h5>
 
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                <h5>APPROXIMATELY</h5>
 
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                <div class="stats__count">70</div>
 
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                <h5>KWH OF POWER ANNUALLY</h5>
 
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                <h5>USED TO POWER SYSTEM</h5>
 
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                <h5>PROVIDES UP TO</h5>
 
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                <div class="stats__count">1700</div>
 
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                <h5>LUX OF LIGHT</h5>
 
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                <h5>TO GROW SEEDS</h5>
 
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                <h5>CONTAINS</h5>
 
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                <div class="stats__count">120</div>
 
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                <h5>INDIVIDUALLY ADDRESSABLE</h5>
 
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                <h5>LOW-POWER LED'S</h5>
 
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                    <p style="font-size:100%">Having identified the design parameters for the system, the next stage was to begin ordering parts and putting it together. The system was divided into three independent, functional sub-systems to make the task of assembling the system more manageable and allowing team members to focus on the sub-system that most suited their specialty. These three sub-systems were hardware, software and biological aspects.</p>
 
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                    <p style="font-size:100%">The function of the hardware is to contain the electronics and organisms, power the LED’s/microcontroller and maximise the light available to the plants. Containment is through the use of a sealed box, with a detachable lid for access. This box is glued with tin foil and sprayed black to minimise exchange of light with the environment. Powering the LED’s proved to be more difficult, taking our engineers many days to find the optimal solution. You can find all the grizzly details on this process here. However, essentially the system is powered from a 5V 2.1A AC adapter that plugs straight in to your mains power supply. Alternatively, you can use 4 AA batteries to power the system for short periods of time if necessary. The LED’s are wired in parallel so the same light is provided along the length of the container. This can be seen from images in the Gallery.</p>
 
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                    <p style="font-size:100%">The purpose of the software is to control the LED’s, by allowing the user to easily adapt features such as light intensity, wavelength and also specify the length of the day/night cycle. For our design, we use the Arduino UNO microcontroller to control these characteristics as it offers a user-friendly interface and is well-suited to our design. You can find all the code laid bare and a guide to the Arduino here.</p>
 
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                    <p style="text-align:center"><br>The engineers, hard at work trying to troubleshoot issues with the system.</p>
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                     <p><font size="3">The United Nations estimates global population has increased by 1 billion over 12 years and will near 9.8 billion by 2050 [4]. This increase has meant social demand has also grown at an unparalleled rate. However, a more pressing issues is that of food security.  Food security is defined as one’s ability to have adequate access to sufficient food. Said food must be safe and nutritious as for an individual to maintain a healthy and active lifestyle. </font></p>
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                    <p style="text-align:center"><br>The finished product, set to a rainbow function that cycles through various wavelengths of light</p>
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                    <p><font size="3">To match demand, the agricultural sector oft utilises synthetic fertilisers to improve crop growth. Nitrogen, phosphate and potassium (NPK) based fertilisers are used as they provide crops with essential macronutrients required for growth. NPK consumption is predicted to increase to 201.7 million tonnes by the end of 2020 [5].  </font></p>
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                    <p><font size="3">Synthetic NPK fertilisers significant increases and yield of popular crops such as maize and soybean (3) but they also play a large role in in climate change. Nitrogenous fertilisers are produced by the Haber-Bosch process. This process is highly energy intensive, requiring 600kg of natural gas to produce 1000kg of ammonium, producing  670 million tonnes of CO2 per annum [6].</font></p>
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                    <p><font size="3">Repeatedly, studies show fertiliser application has a negative long-term impact on soil health. Synthetic fertilisers cause soil pH to decrease which degrades soil crumbs. This results in compact soil with reduced water drainage and air circulation; both of which have negative impacts on plant-root health [7].</font></p>   
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                     <p><font size="3">This is not the only local impact of synthetic fertilisers. Accumulation of plant nutrients in bodies of water, resulting from surface run-off, leads to eutrophication. Eutrophication, from Greek meaning ‘well-nourished’, impacts water quality and allows algal blooming [8]. Algal blooming can impact biodiversity through toxin production and promotion of a hypoxic environment (Figure 1)[9]</font></p> 
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                    <p><font size="3">Eutrophication may have a larger humanitarian impact in the future. Eutrophication causes an increase in grey water percentage; the polluted water associated with industry. If grey water is not processed, then clean drinking water availability is reduced. This will likely impact rural areas of less economically developed countries where money and infrastructure is not readily available to cover water processing costs.</font></p> 
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                    <p><font size="3">Residents are forced to either drink unsafe water or travel further to access water that is safer, however, these oases may become even rarer as water sources become exhausted or contaminated. This issue becomes more concerning when considering that 48% of the total population of Africa relies upon agriculture [10], a continent that experienced a 10.6% increase in total fertiliser demand between 2012 and 2018 [11].</font></p>
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                    <p><font size="3">These issues are expected to become more widespread in the future, with the aforementioned population increase and current demands for agriculture produce putting more pressure on farmers to maintain, if not increase, usage of synthetic fertilisers. As such, to overcome risks moving into the future, research into more sustainable nitrogen fertilisers and their substitutes should be pursued.</font></p>
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<h3 class="subhead">Description</h3>
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                 <h1 class="display-2">Solution</h1>
 
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                    <p><font size="3">An effective solution would overcome both the energy expenditure and pollution associated with inorganic fertilisers but without losing properties such as broad and simple usage. Therefore our solution aims to create a single-application, broad host range sustainable alternative in the form of an engineered root endophyte that acts as a microbial adapter. This endophyte is Pseudomonas fluorescens.</font></p>
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                    <p><font size="3">A gram-negative bacterium with a diverse metabolism, P. fluorescens has been highlighted as a diverse plant growth promoting bacterium [12] capable of colonising a broad range of plant roots [13].</font></p>
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                    <p><font size="3">Pseudomonas fluorescens is known as a natural plant growth promoter for numerous reasons;</font></p>
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<li>It produces a siderophore that liberates iron [14], consequentially liberating phosphorus too.[15]</li>
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<li>It has anti-fungal properties (protecting from pathogens).[16]</li>
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<li>It is nematophagous and produces nematode/protozoa repellents, protecting from parasites. [17]</li>
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<li>Produces anti-insectal toxins, protecting from pests. [18]</li>
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<li>Induces systemic resistance and tolerance.[19]</li>
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<p><font size="3">With all these features, pseudomonas fluorescens is already an ideal organism for improving crop yields, but the Newcastle iGEM project takes this a step further.</font></p>
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<p><font size="3">By engineering P. fluorescens to express novel genes the team aims to manipulate the soil microbial community via chemical attraction/repulsion to achieve desired processes. In our case this is a nutrient sustaining soil but there are no limits! From soil remediation to pest control, this project aims to create a chassis out of Pseudomonas fluorescens so future scientists can manipulate the soil community in any way they like.</font></p>
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<p><font size="3">Our prototype focuses on sustaining the amount of Nitrogen present in soils without adding fertiliser or causing run-off. To combat this, we have introduced flavonoid biosynthesis genes to Pseudomonas fluorescens that attract free-living/non-nodulating nitrogen fixing bacteria to improve the nitrogen content of the soil [20].</font></p>
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<p><font size="3">This method means that one application is all that is needed to improve the nutrient availability for a plants life-time. This combined with the other protective roles of P. fluorescens acts to improve crop yields without genetically modifying plants and without Nitrogen/Phosphorus fertilisers. Even if we only reduce fertiliser use by a tiny amount, globally this would make a huge difference in terms of energy usage and pollution.</font></p>
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                            <h3 class=><font color="white">GROWING IN URBAN SPACES</font></h3>
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                    <h1 class="display-2 display-2--light">TEST</h1>
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                <h1 class="display-2"><font color="white">Advantages of Contained Agriculture</font></h1>
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                    <font size="4" font face="verdana" font color="green">Substantial time was spent carrying out extensive research, both inside and outside the lab, in order to optimise the system for the target audience. This included speaking with organisations and individuals in industry who are involved with hydroponics-based systems or those who may be interested in working with such a system in the future. Some of the individuals we liaised with include Chris Tapsell, the Research Director of KWS UK, one of the biggest seed companies in the world, and Richard Ballard, co-founder of Growing Underground in London where they hydroponically grow micro greens and salad leaves 33 metres below the ground. These potential clients helped us focus our product so that it can better meet the needs of our clients.</font><br><br>
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                    <font size="4" font face="verdana" font color="green">In addition to gathering external opinion on our system, we also did our own tests on system performance. This included tests to verify the optimal light intensity, wavelength and positioning. The graph below illustrates how the light intensity (measured in lux) varies over time (in seconds) when the system is operated under various wavelengths of light. The black line indicates the system running with the rainbow function loaded which cyclically varies the light wavelength. As the results showed that blue, red and purple light and provided the most lux we are currently using these in the system but plan to use the rainbow function too in future to see how this affects growth or the aesthetics of the plant.
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                  <p><font size="3">The effects of climate change are becoming more noticeable as time progresses; we are losing staggering amounts of valuable farmland due to mass flooding, freak weather events, soil erosion, infectious diseases and deforestation. Over the next 50 years, farming is going to become even more marginalised [21].</p>
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                    <p><font size="3">One way of protecting our crops and the land we use for agriculture is by growing within controlled, contained environments. Growing indoors is already a well-established practice; greenhouses are widely used and guarantee a safer, and more predictable method of growing all year round. There are many benefits of applying the contained, controlled environments found in greenhouses into urban spaces, these include:</p>
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<li>Providing cities with fresh produce all year round.</li>
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                    <p><font size="3"><br>With developing technologies in the field of sustainable energy, it could one day be possible to engineer contained growth systems that are self-sustaining in regards to its energy usage. By carefully controlling the parameters within these environments, we are able to emulate perfect surroundings that allow the crops to grow to their full potential, maximising yield.</p>
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                    <p><font size="3">Our project plans to use genetically modified bacteria, which means we will be working with GMO’s, but what are GMO’s? - “Genetically modified organisms (GMOs) can be defined as organisms (i.e. plants, animals or microorganisms) in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination.”[22]</p>
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                    <p><font size="3">Integrations of GMO’s into the natural environment pose many concerns to both science and ecological communities. Introducing gm crops into the wild holds the potential to introduce engineered genes into foreign species. The effects of GMO release are widely unidentified, this is the main area of concern as there so many unknowns.</p>
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                    <p><font size="3">The use of GM bacteria means that we have to take precautions when integrating it into the real world. We have identified the ways to ensure systems are enclosed and risk of GM run-off is minimised on our  <a href="https://2018.igem.org/Team:Newcastle/Safety" class="white">Safety Page</a>.</p>
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<p class="about-para"><font size="2">1. Jousset, A., et al. (2009). "Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters." The Isme Journal 3: 666<font></p>
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<p class="about-para"><font size="2">2. Jousset, A., et al. (2009). "Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters." The Isme Journal 3: 666<font></p>
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<p class="about-para"><font size="2">3. Vanitha SC & Umesha S (2011) Pseudomonas fluorescens mediated systemic resistance in tomato is driven through an elevated synthesis of defense enzymes. Biologia Plantarum 55(2):317-322.<font></p>
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<p class="about-para"><font size="2">4. United Nations, Department of Economic and Social Affairs, Population Division (2017) World Population Prospects: The 2017 Revision, Key Findings and Advance Tables. https://population.un.org/wpp/Publications/Files/WPP2017_KeyFindings.pdf<font></p>
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<p class="about-para"><font size="2">5. Food and Agriculture Organization of the United Nations (2015) World Fertilizer Trends and Outlook to 2018. http://www.fao.org/3/a-i4324e.pdf<font></p>
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<p class="about-para"><font size="2">6.  Usman MN, MG; Musa, I (2015) Effect of Three Levels of NPK Fertilizer on Growth Parameters and Yield of Maize-Soybean Intercrop. International Journal of Scientific and Research Publications 5(9).<font></p>
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<p class="about-para"><font size="2">7. Pfromm PH (2017) Towards sustainable agriculture: Fossil-free ammonia. Journal of Renewable and Sustainable Energy 9(3):034702.<font></p>
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<p class="about-para"><font size="2">8. Bitew YA, M (2017) Impact of Crop Production Inputs on Soil Health: A Review. Asian Journal of Plant Sciences 16(3):109-131.<font></p>
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<p class="about-para"><font size="2">9. Yang X-e, Wu X, Hao H-l, & He Z-l (2008) Mechanisms and assessment of water eutrophication. Journal of Zhejiang University. Science. B 9(3):197-209.<font></p>
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<p class="about-para"><font size="2">10. Carmichael WW (2001) Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”. Human and Ecological Risk Assessment: An International Journal 7(5):1393-1407.<font></p>
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<p class="about-para"><font size="2">11. New Partnership for Africa's Development (2013) Agriculture in Africa - Transformation and Outlook. http://www.un.org/en/africa/osaa/pdf/pubs/2013africanagricultures.pdf<font></p>
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<p class="about-para"><font size="2">12. Food and Agriculture Organization of the United Nations (2017) World Fertilizer Trends and Outlook to 2020. http://www.fao.org/3/a-i6895e.pdf<font></p>
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<p class="about-para"><font size="2">13. Bergey, D. H., et al. (1984). Bergey's manual of systematic bacteriology. Baltimore, MD, Williams & Wilkins.<font></p>
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<p class="about-para"><font size="2">14. Gómez-Lama Cabanás C, Schilirò E, Valverde-Corredor A, & Mercado-Blanco J (2014) The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots. Frontiers in Microbiology 5:427.<font></p>
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<p class="about-para"><font size="2">15. Gross, H. and J. Loper (2009). Genomics of secondary metabolite production by Pseudomonas spp.<font></p>
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<p class="about-para"><font size="2">16. Sharma SB, Sayyed RZ, Trivedi MH, & Gobi TA (2013) Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2:587.<font></p>
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<p class="about-para"><font size="2">17. Ruffner, B., et al. (2013). "Oral insecticidal activity of plant-associated pseudomonads." Environmental Microbiology 15(3): 751-763.<font></p>
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<p class="about-para"><font size="2">18. Jousset, A., et al. (2009). "Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters." The Isme Journal 3: 666<font></p>
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<p class="about-para"><font size="2">19. Vanitha SC & Umesha S (2011) Pseudomonas fluorescens mediated systemic resistance in tomato is driven through an elevated synthesis of defense enzymes. Biologia Plantarum 55(2):317-322.<font></p>
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<p class="about-para"><font size="2">20. Maheshwari DK (2012) Bacteria in Agrobiology: Plant Probiotics (Springer Berlin Heidelberg).<font></p>
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<p class="about-para"><font size="2">21. Despommier D (2011) The vertical farm: Controlled environment agriculture carried out in tall buildings would create greater food safety and security for large urban populations. J fur Verbraucherschutz und Leb 6(2):233–236.<font></p>
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<p class="about-para"><font size="2">22.World Health Organization. (2018). Q&A: genetically modified food. [online] Available at: http://www.who.int/foodsafety/areas_work/food-technology/faq-genetically-modified-food/en/ [Accessed 13 Sep. 2018].<font></p>
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Alternative Roots

InterLab Study

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2018 Interlab study aims

Problems With Fertiliser

The United Nations estimates global population has increased by 1 billion over 12 years and will near 9.8 billion by 2050 [4]. This increase has meant social demand has also grown at an unparalleled rate. However, a more pressing issues is that of food security. Food security is defined as one’s ability to have adequate access to sufficient food. Said food must be safe and nutritious as for an individual to maintain a healthy and active lifestyle.

To match demand, the agricultural sector oft utilises synthetic fertilisers to improve crop growth. Nitrogen, phosphate and potassium (NPK) based fertilisers are used as they provide crops with essential macronutrients required for growth. NPK consumption is predicted to increase to 201.7 million tonnes by the end of 2020 [5].

Synthetic NPK fertilisers significant increases and yield of popular crops such as maize and soybean (3) but they also play a large role in in climate change. Nitrogenous fertilisers are produced by the Haber-Bosch process. This process is highly energy intensive, requiring 600kg of natural gas to produce 1000kg of ammonium, producing 670 million tonnes of CO2 per annum [6].

Repeatedly, studies show fertiliser application has a negative long-term impact on soil health. Synthetic fertilisers cause soil pH to decrease which degrades soil crumbs. This results in compact soil with reduced water drainage and air circulation; both of which have negative impacts on plant-root health [7].

This is not the only local impact of synthetic fertilisers. Accumulation of plant nutrients in bodies of water, resulting from surface run-off, leads to eutrophication. Eutrophication, from Greek meaning ‘well-nourished’, impacts water quality and allows algal blooming [8]. Algal blooming can impact biodiversity through toxin production and promotion of a hypoxic environment (Figure 1)[9]

Eutrophication may have a larger humanitarian impact in the future. Eutrophication causes an increase in grey water percentage; the polluted water associated with industry. If grey water is not processed, then clean drinking water availability is reduced. This will likely impact rural areas of less economically developed countries where money and infrastructure is not readily available to cover water processing costs.

Residents are forced to either drink unsafe water or travel further to access water that is safer, however, these oases may become even rarer as water sources become exhausted or contaminated. This issue becomes more concerning when considering that 48% of the total population of Africa relies upon agriculture [10], a continent that experienced a 10.6% increase in total fertiliser demand between 2012 and 2018 [11].

These issues are expected to become more widespread in the future, with the aforementioned population increase and current demands for agriculture produce putting more pressure on farmers to maintain, if not increase, usage of synthetic fertilisers. As such, to overcome risks moving into the future, research into more sustainable nitrogen fertilisers and their substitutes should be pursued.





Description

Solution

An effective solution would overcome both the energy expenditure and pollution associated with inorganic fertilisers but without losing properties such as broad and simple usage. Therefore our solution aims to create a single-application, broad host range sustainable alternative in the form of an engineered root endophyte that acts as a microbial adapter. This endophyte is Pseudomonas fluorescens.

A gram-negative bacterium with a diverse metabolism, P. fluorescens has been highlighted as a diverse plant growth promoting bacterium [12] capable of colonising a broad range of plant roots [13].

Pseudomonas fluorescens is known as a natural plant growth promoter for numerous reasons;

  • It produces a siderophore that liberates iron [14], consequentially liberating phosphorus too.[15]
  • It has anti-fungal properties (protecting from pathogens).[16]
  • It is nematophagous and produces nematode/protozoa repellents, protecting from parasites. [17]
  • Produces anti-insectal toxins, protecting from pests. [18]
  • Induces systemic resistance and tolerance.[19]

With all these features, pseudomonas fluorescens is already an ideal organism for improving crop yields, but the Newcastle iGEM project takes this a step further.

By engineering P. fluorescens to express novel genes the team aims to manipulate the soil microbial community via chemical attraction/repulsion to achieve desired processes. In our case this is a nutrient sustaining soil but there are no limits! From soil remediation to pest control, this project aims to create a chassis out of Pseudomonas fluorescens so future scientists can manipulate the soil community in any way they like.

Our prototype focuses on sustaining the amount of Nitrogen present in soils without adding fertiliser or causing run-off. To combat this, we have introduced flavonoid biosynthesis genes to Pseudomonas fluorescens that attract free-living/non-nodulating nitrogen fixing bacteria to improve the nitrogen content of the soil [20].

This method means that one application is all that is needed to improve the nutrient availability for a plants life-time. This combined with the other protective roles of P. fluorescens acts to improve crop yields without genetically modifying plants and without Nitrogen/Phosphorus fertilisers. Even if we only reduce fertiliser use by a tiny amount, globally this would make a huge difference in terms of energy usage and pollution.

GROWING IN URBAN SPACES

Advantages of Contained Agriculture

The effects of climate change are becoming more noticeable as time progresses; we are losing staggering amounts of valuable farmland due to mass flooding, freak weather events, soil erosion, infectious diseases and deforestation. Over the next 50 years, farming is going to become even more marginalised [21].

One way of protecting our crops and the land we use for agriculture is by growing within controlled, contained environments. Growing indoors is already a well-established practice; greenhouses are widely used and guarantee a safer, and more predictable method of growing all year round. There are many benefits of applying the contained, controlled environments found in greenhouses into urban spaces, these include:

  • Providing cities with fresh produce all year round.
  • Reducing the Carbon footprint of crop production due to reduced food millage.
  • No agricultural run-off.
  • Limited need for pesticides and herbicides.
  • Safer crops as there is less risk of contamination.
  • Reduced spoilage because of shorter transportation times and reduced handling.
  • Less agricultural pollution.


With developing technologies in the field of sustainable energy, it could one day be possible to engineer contained growth systems that are self-sustaining in regards to its energy usage. By carefully controlling the parameters within these environments, we are able to emulate perfect surroundings that allow the crops to grow to their full potential, maximising yield.

Our project plans to use genetically modified bacteria, which means we will be working with GMO’s, but what are GMO’s? - “Genetically modified organisms (GMOs) can be defined as organisms (i.e. plants, animals or microorganisms) in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination.”[22]

Integrations of GMO’s into the natural environment pose many concerns to both science and ecological communities. Introducing gm crops into the wild holds the potential to introduce engineered genes into foreign species. The effects of GMO release are widely unidentified, this is the main area of concern as there so many unknowns.

The use of GM bacteria means that we have to take precautions when integrating it into the real world. We have identified the ways to ensure systems are enclosed and risk of GM run-off is minimised on our Safety Page.





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REFERENCES

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