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                 <h1 class="display-2">Project Overview</h1>
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                 <h1 class="display-2">Environmental Imperative</h1>
 
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                    <p><font size="3">The United Nations estimates global population has increased by over 1 billion since 2005 and will near 9.8 billion by 2050 [1]. The demand that we place on agricultural products rises in parallel. We rely on the agricultural sector to provide not only food, but also fuels, shelter and fabrics. In turn, the agricultural sector relies on the application of synthetic fertilisers to maintain crop productivity. Nitrogen, phosphate and potassium (NPK) based fertilisers provide crops with essential macronutrients required for growth. NPK consumption is predicted to increase to 201.7 million tonnes by the end of 2020 [2]. </font></p>
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<p><font size="3"><p><font size="3">NPK fertilisers significantly increase the yield of crops such as maize, wheat and potatoes [3] but they also play a large role in climate change. Nitrogenous fertilisers are produced using the Haber-Bosch process. This process is energy intensive, requiring 600 kg of natural gas to produce 1000 kg of ammonium, and resulting in the release of 670 million tonnes of CO<sub>2</sub> per annum [4].</font></p>
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                        <div class="text">Soils contain diverse microbial communities</div>
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                     <p><font size="3">Fertiliser application has also been shown to have a negative long-term impact on soil health. Synthetic fertilisers cause soil pH to decrease, degrading soil crumbs and resulting in compact soils with reduced water drainage and air circulation; both have negative impacts on plant-root health [5]. Meanwhile, the accumulation of fertiliser run-off leads to eutrophication in water courses. Eutrophication impacts water quality and allows algal blooms to form, affecting biodiversity through toxin production and promotion of a hypoxic environment [6,7]. This reduces the availability of clean drinking water with additional costs incurred to process water for drinking.</font></p>
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                        <div class="text">Within these communities are microbes with useful properties</div>
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                        <div class="text">Endophytes are microbes that live harmlessly within plant tissues</div>
 
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                        <div class="text">Can we programme endophytes to influence the wider microbial community?</div>
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                        <div class="text">Could they synthesise chemicals to attract beneficial soil microbes?</div>
 
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                        <div class="text">Attracting bacteria to fix nitrogen and reducing the need for chemical fertilisers</div>
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                        <div class="text">Or maybe the endophytes can synthesise chemicals that deter pests or pathogens?</div>
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                        <div class="text">Alternative Roots: engineering endophytes for smart agricultural solutions</div>
 
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                <h1 class="display-2"><font color="white">Symbiosis</font></h1>
 
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                  <p><font size="3">A plant’s demand for resources long predates the era of fertilisers, this demand has led to co-evolution of symbiotic relationships between plants and microbes. The legume-rhizobia symbiosis is one of the most well-known of these interactions. These bacteria are able to colonise the roots of plants such as peas where they fix atmospheric nitrogen. This nitrogen is then readily available for the plant to access [8]. </p>
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<img src="https://static.igem.org/mediawiki/2018/6/64/T--Newcastle--RhizobiainRootTom.png"><p><font size="2"><center>Figure 1. <i>Rhizobia</i> spp. colonising the root of broad bean plant in Northumberland, UK. Photo credit: Dr Thomas Howard</center></font></p>
  
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                    <p><font size="3">However, it is not just bacteria that plants have evolved these beneficial relationships with. Mycorrhizal fungi  are capable of producing  intricate hyphal networks throughout the root system that enhance the plant’s ability to access nutrients and water [9]. </p>
  
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<img src="https://static.igem.org/mediawiki/2018/0/03/T--Newcastle--Glomites.jpeg"><p><font size="2"><center>Figure 2. <i>Glomites rhyniensis</i> hyphae growing through the root cortex of <i>Aglophyton major</i> [10]</center></font></p>
  
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                  <p><font size="3">Plant interactions with soil micro-organisms are nature’s wide-ranging alternative to chemical fertilisers. These natural symbioses date back over 400 million years to the Devonian era, where fossil records show evidence that even the earliest land plants had relationships with fungal endophytes [10]. </p>
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                    <p><font size="3">Engineering of these symbioses, to open access to crop plants, is a promising solution to aid in mitigating NPK fertiliser dependence. While there have been many attempts to engineer nitrogen fixation into the crop itself, the concept of engineering symbioses themselves is a relatively untouched area. Here we propose an alternative route to reach this goal. Rather than engineering the plant or nitrogen-fixing symbiosis directly, we instead put forward the idea to develop a novel endophytic bacterial chassis to act as a mediator between these beneficial bacteria and the plant roots. </p>
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                 <h1 class="display-2">Introduction</h1>
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                     <p><font size="3">Sustainability is a topic of increasing concern in the field of agriculture, food security and rural development. There is a dire need for innovation in this field; primarily driven by predictions of substantial global population increase coupled with severe pressure on non-renewable resources. The result is a necessity to increase food production whilst reducing our impact on the environment. As such, our aim is to find sustainable solutions that address some of these issues. </font></p>
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                     <p><font size="3">This project investigated <i>Pseudomonas</i> sp. (CT 364) due to evidence that members of this genus are capable of colonising plant roots [11]. <i>Pseudomonas</i> are Gram-negative bacteria with a diverse metabolism [12] that enables the reported colonisation of a broad range of plant roots. Because of this, several <i>Pseudomonas</i> species have been proposed as natural plant growth promoters due to their capacity to produce siderophores which are able to liberate iron [13] and phosphorus [14]. The genus has also been implicated with the production of anti-fungal chemicals [15] and nematode repellents [16]. </font></p>
  
                    <p><font size="3">Inorganic nitrogen fertiliser production is extremely energy-intensive, accounting for ~1 % of all global energy use.[1] These fertilisers require persistent application as they are rapidly leeched out of the soil shortly after application causing mass pollution worldwide. </font></p>
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<p><font size="3"><p><font size="3">Considering these benefits, Alternative Roots investigated whether it would be possible to develop <i>Pseudomonas</i> sp. as the first synthetic biology ready endophytic bacterium.</font></p>
  
                    <p><font size="3">The problems with inorganic fertilisers are that they do not last long, they take masses of energy to produce and cause pollution, the most common fertilisers are nitrogen, phosphorus and potassium, the Newcastle iGEM 2018 team aim to provide a source of these nutrients that is sustainable and non-polluting.</font></p>
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                 <h1 class="display-2">Introducing <i>Pseudomonas</i> sp.</h1>
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                     <p><font size="3">The organism used by the team is a Gram-negative bacterium called <i>Pseudomonas</i> sp. <i>Pseudomonas</i> sp. lives in soil and water, and is capable of colonising roots. Naturally <i>Pseudomonas</i> sp. is known as a plant growth promoter for multiple reasons; </font></p>
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                     <p><font size="3">In addition to examining the synthetic biology of this proposal we have also examined potential deployment options. Examination of urban agricultural production led us to concepts such as urban farming and contained agriculture. These offer benefits to this proposal as they offer a route to containing any GMOs, as well as many other advantages: </font></p>
  
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<li>It produces a siderophore that liberates iron [2], consequentially liberating phosphorus too. [3]</li>
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<li><font size="3">Producing locally-grown, fresh produce all year round.</font></li>
<li>It has anti-fungal properties (protecting from pathogens). [4]</li>
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<li><font size="3">Reducing the carbon footprint of crop production due to reduced food millage.</font></li>
<li>It is nematophagous, protecting plants from parasitic nematode worms. [5]</li>
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<li><font size="3">No agricultural run-off.</font></li>
<li>Produces anti-insectal toxins, protecting from pests. [6]</li>
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<li><font size="3">Limited need for pesticides and fertilisers.</font></li>
<li>It is thought to induce systemic resistance and/or tolerance. [7]</li>
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<li><font size="3">Safer crops as there is less risk of contamination.</font></li>
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<li><font size="3">Reduced spoilage because of shorter transportation times and reduced handling.</font></li>
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<p><font size="3">With developing technologies in the field of sustainable energy, it could one day be possible to engineer contained growth systems that are self-sustaining regarding its energy usage. By carefully controlling the parameters within these environments, we can emulate perfect surroundings that allow the crops to grow to their full potential, maximising yield.</font></p>
  
                  <p><font size="3"> With all these features,<i>Pseudomonas</i> sp. was already an ideal organism for improving crop yields, but the Newcastle iGEM team wanted to take this is a step further.</font></p>
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                    <p><font size="3">We are attempting to use a system like this in our project. Newcastle City council has recently declared their bold plans to convert Newcastle into a ‘Smart City.’ Looking into the proposed scenarios, we saw an opportunity to propose a ‘sustainable agriculture’ scenario for Newcastle, incorporating our hardware development. To do this we have put together a theoretical design project that outlines how we may implement this idea in Newcastle. </font></p>
       
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                  <p><font size="3">By engineering <i>Pseudomonas</i> sp. 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 an engineerable chassis out of <i>Pseudomonas</i> sp. so future scientists can manipulate the soil community in any way they like.</p>
 
 
                    <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 flavonoids to <i>Pseudomonas</i> sp. that attract free-living/non-nodulating nitrogen fixing bacteria to improve the nitrogen content of the soil.</p>
 
 
                    <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 <i>Pseudomonas</i> sp. 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. </p>
 
 
 
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                     <p><font size="3">Sustainability is a topic of increasing concern in the fields of agriculture, food security and rural development. There is a dire need for innovation in this field; primarily driven by predictions of substantial global population increase coupled with severe pressure on non-renewable resources. The result is a necessity to increase food production whilst reducing our impact on the environment. As such, our aim is to find sustainable solutions that address some of these issues. </font></p>
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                     <p><font size="3">Our goal was to characterise the endophytic properties of <i>Pseudomonas</i> sp., our proposed endophytic chassis, and to establish transformation protocols. <i>Pseudomonas</i> sp. could subsequently be engineered to express genes of interest resulting in plant physiological changes when colonising plant roots.</font></p>
  
                     <p><font size="3">Inorganic nitrogen fertiliser production is extremely energy-intensive, accounting for ~1 % of all global energy use (Smith 2002  http://science.sciencemag.org/content/297/5587/1654.long ). These fertilisers require persistent application as they are rapidly leeched out of the soil shortly after application causing mass pollution worldwide. </font></p>
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                     <p><font size="3">The goal of our Human Practices work was to generate a conversation locally about the use of Newcastle's Victoria Tunnel as an urban farm. In doing so, we wanted to raise larger questions surrounding the use of GM bacteria to increase plant productivity, as opposed to genetically modifying the plant itself - challenging consumer views on GMOs.</font></p>
  
                     <p><font size="3">The problems with inorganic fertilisers are that they do not last long, they take masses of energy to produce and cause pollution, the most common fertilisers are Nitrogen, phosphorus and potassium, the Newcastle 2018 team aim to provide a source of these nutrients that is sustainable and non-polluting.</font></p>
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                     <p><font size="3">Alongside this, our goal was to build a hardware prototype for the Victoria Tunnel; and address the lack of suitable hardware that would allow us to grow large numbers of plant seedlings in a controlled environment for the purposes of our project. The hardware needed to be cheap, programmable, energy and cost efficient, and a standardised method for growing plants.</font></p>
 
        
 
        
  
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<h3 class="subhead">Description</h3>
 
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                 <h1 class="display-2">References & Attributions</h1>
 
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<p class="about-para"><font size="2"><strong>Attributions: Connor Trotter, Will Tankard, Chris Carty, Frank Eardley, Heather Bottomley, Lewis Tomlinson, Luke Waller, Patrycja Ubysz, Sadiya Quazi, and Umar Farooq.</strong><font></p>
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 +
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<p class="about-para"><font size="2">1. United Nations, Department of Economic and Social Affairs., Population Division (2017). "World Population Prospects: The 2017 Revision, Key Findings and Advance Tables." <font></p>
 +
 +
 +
<p class="about-para"><font size="2">2. United Nations Food and Agriculture Organization (2017) World Fertilizer Trends and Outlook to 2020. <font></p>
 +
 +
<p class="about-para"><font size="2">3. 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>
 +
 +
<p class="about-para"><font size="2">4. Pfromm PH (2017) Towards sustainable agriculture: Fossil-free ammonia. Journal of Renewable and Sustainable Energy 9(3):034702. <font></p>
  
 +
<p class="about-para"><font size="2">5. 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>
  
 +
<p class="about-para"><font size="2">6. 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>
  
 +
<p class="about-para"><font size="2">7. 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>
  
 +
<p class="about-para"><font size="2">8. Zahran HH (1999) Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Conditions and in an Arid Climate. Microbiology and Molecular Biology Reviews 63(4):968-989. <font></p>
  
 +
<p class="about-para"><font size="2">9. Birhane E, Kuyper TW, Sterck FJ, Gebrehiwot K, & Bongers F (2015) Arbuscular mycorrhiza and water and nutrient supply differently impact seedling performance of dry woodland species with different acquisition strategies. Plant Ecology & Diversity 8(3):387-399.<font></p>
  
 +
<p class="about-para"><font size="2">10. Taylor TN, Remy W, Hass H, & Kerp H (1995) Fossil Arbuscular Mycorrhizae from the Early Devonian. Mycologia 87(4):560-573.<font></p>
  
 +
<p class="about-para"><font size="2">11. Buddrus-Schiemann K, Schmid M, Schreiner K, Welzl G, & Hartmann A (2010) Root Colonization by Pseudomonas sp. DSMZ 13134 and Impact on the Indigenous Rhizosphere Bacterial Community of Barley. Microbial Ecology 60(2):381-393.<font></p>
  
 +
<p class="about-para"><font size="2">12. Bergey DH, Krieg NR, & Holt JG (1984) Bergey's manual of systematic bacteriology (Williams & Wilkins, Baltimore, MD). <font></p>
  
 +
<p class="about-para"><font size="2">13. Sah S, Singh N, & Singh R (2017) Iron acquisition in maize (Zea mays L.) using Pseudomonas siderophore. 3 Biotech 7(2):121. <font></p>
  
 +
<p class="about-para"><font size="2">14. 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>
  
 +
<p class="about-para"><font size="2">15. Ruffner B, et al. (2013) Oral insecticidal activity of plant-associated pseudomonads. Environmental microbiology 15(3):751-763.<font></p>
  
<p class="about-para"><font size="2">1. Smith. B (2002). "Nitrogenase Reveals Its Inner Secrets" Science Journal 297: 5587<font></p>
+
<p class="about-para"><font size="2">16. Jousset A, et al. (2009) Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters. The ISME journal 3(6):666-674. <font></p>
  
<p class="about-para"><font size="2">2. Gómez-Lama Cabanás C, Schilirò E, Valverde-Corredor A, & Mercado-Blanco J (2014) The Biocontrol Endophytic Bacterium <i>Pseudomonas fluorescens</i> PICF7 Induces Systemic Defense Responses in Aerial Tissues Upon Colonization of Olive Roots. Frontiers in Microbiology 5:427.<font></p>
+
<p class="about-para"><font size="2">17. 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.</p>
  
<p class="about-para"><font size="2">3. Gross, H. and J. Loper (2009). Genomics of Secondary Metabolite Production by <i>Pseudomonas</i> spp.<font></p>
+
<p class="about-para"><font size="2">18. 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.</p>
  
<p class="about-para"><font size="2">4. 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>
 
  
<p class="about-para"><font size="2">5. Ruffner, B., et al. (2013). "Oral Insecticidal Activity of Plant-Associated Pseudomonads." Environmental Microbiology 15(3): 751-763.<font></p>
 
  
<p class="about-para"><font size="2">6. 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>
 
  
<p class="about-para"><font size="2">7. 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>
 
  
  

Latest revision as of 23:46, 17 October 2018

Alternative Roots

Alternative Roots

Project Description




Environmental Imperative

The United Nations estimates global population has increased by over 1 billion since 2005 and will near 9.8 billion by 2050 [1]. The demand that we place on agricultural products rises in parallel. We rely on the agricultural sector to provide not only food, but also fuels, shelter and fabrics. In turn, the agricultural sector relies on the application of synthetic fertilisers to maintain crop productivity. Nitrogen, phosphate and potassium (NPK) based fertilisers provide crops with essential macronutrients required for growth. NPK consumption is predicted to increase to 201.7 million tonnes by the end of 2020 [2].

NPK fertilisers significantly increase the yield of crops such as maize, wheat and potatoes [3] but they also play a large role in climate change. Nitrogenous fertilisers are produced using the Haber-Bosch process. This process is energy intensive, requiring 600 kg of natural gas to produce 1000 kg of ammonium, and resulting in the release of 670 million tonnes of CO2 per annum [4].

Fertiliser application has also been shown to have a negative long-term impact on soil health. Synthetic fertilisers cause soil pH to decrease, degrading soil crumbs and resulting in compact soils with reduced water drainage and air circulation; both have negative impacts on plant-root health [5]. Meanwhile, the accumulation of fertiliser run-off leads to eutrophication in water courses. Eutrophication impacts water quality and allows algal blooms to form, affecting biodiversity through toxin production and promotion of a hypoxic environment [6,7]. This reduces the availability of clean drinking water with additional costs incurred to process water for drinking.



Symbiosis

A plant’s demand for resources long predates the era of fertilisers, this demand has led to co-evolution of symbiotic relationships between plants and microbes. The legume-rhizobia symbiosis is one of the most well-known of these interactions. These bacteria are able to colonise the roots of plants such as peas where they fix atmospheric nitrogen. This nitrogen is then readily available for the plant to access [8].

Figure 1. Rhizobia spp. colonising the root of broad bean plant in Northumberland, UK. Photo credit: Dr Thomas Howard

However, it is not just bacteria that plants have evolved these beneficial relationships with. Mycorrhizal fungi  are capable of producing  intricate hyphal networks throughout the root system that enhance the plant’s ability to access nutrients and water [9].

Figure 2. Glomites rhyniensis hyphae growing through the root cortex of Aglophyton major [10]

Plant interactions with soil micro-organisms are nature’s wide-ranging alternative to chemical fertilisers. These natural symbioses date back over 400 million years to the Devonian era, where fossil records show evidence that even the earliest land plants had relationships with fungal endophytes [10]. 

Engineering of these symbioses, to open access to crop plants, is a promising solution to aid in mitigating NPK fertiliser dependence. While there have been many attempts to engineer nitrogen fixation into the crop itself, the concept of engineering symbioses themselves is a relatively untouched area. Here we propose an alternative route to reach this goal. Rather than engineering the plant or nitrogen-fixing symbiosis directly, we instead put forward the idea to develop a novel endophytic bacterial chassis to act as a mediator between these beneficial bacteria and the plant roots.




A New Chassis

This project investigated Pseudomonas sp. (CT 364) due to evidence that members of this genus are capable of colonising plant roots [11]. Pseudomonas are Gram-negative bacteria with a diverse metabolism [12] that enables the reported colonisation of a broad range of plant roots. Because of this, several Pseudomonas species have been proposed as natural plant growth promoters due to their capacity to produce siderophores which are able to liberate iron [13] and phosphorus [14]. The genus has also been implicated with the production of anti-fungal chemicals [15] and nematode repellents [16].

Considering these benefits, Alternative Roots investigated whether it would be possible to develop Pseudomonas sp. as the first synthetic biology ready endophytic bacterium.






Urban Farming

In addition to examining the synthetic biology of this proposal we have also examined potential deployment options. Examination of urban agricultural production led us to concepts such as urban farming and contained agriculture. These offer benefits to this proposal as they offer a route to containing any GMOs, as well as many other advantages:

  • Producing locally-grown, 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 fertilisers.
  • Safer crops as there is less risk of contamination.
  • Reduced spoilage because of shorter transportation times and reduced handling.


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

We are attempting to use a system like this in our project. Newcastle City council has recently declared their bold plans to convert Newcastle into a ‘Smart City.’ Looking into the proposed scenarios, we saw an opportunity to propose a ‘sustainable agriculture’ scenario for Newcastle, incorporating our hardware development. To do this we have put together a theoretical design project that outlines how we may implement this idea in Newcastle.




Alternative Roots

Our goal was to characterise the endophytic properties of Pseudomonas sp., our proposed endophytic chassis, and to establish transformation protocols. Pseudomonas sp. could subsequently be engineered to express genes of interest resulting in plant physiological changes when colonising plant roots.

The goal of our Human Practices work was to generate a conversation locally about the use of Newcastle's Victoria Tunnel as an urban farm. In doing so, we wanted to raise larger questions surrounding the use of GM bacteria to increase plant productivity, as opposed to genetically modifying the plant itself - challenging consumer views on GMOs.

Alongside this, our goal was to build a hardware prototype for the Victoria Tunnel; and address the lack of suitable hardware that would allow us to grow large numbers of plant seedlings in a controlled environment for the purposes of our project. The hardware needed to be cheap, programmable, energy and cost efficient, and a standardised method for growing plants.





Description

References & Attributions

Attributions: Connor Trotter, Will Tankard, Chris Carty, Frank Eardley, Heather Bottomley, Lewis Tomlinson, Luke Waller, Patrycja Ubysz, Sadiya Quazi, and Umar Farooq.

1. United Nations, Department of Economic and Social Affairs., Population Division (2017). "World Population Prospects: The 2017 Revision, Key Findings and Advance Tables."

2. United Nations Food and Agriculture Organization (2017) World Fertilizer Trends and Outlook to 2020.

3. 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).

4. Pfromm PH (2017) Towards sustainable agriculture: Fossil-free ammonia. Journal of Renewable and Sustainable Energy 9(3):034702.

5. Bitew YA, M (2017) Impact of Crop Production Inputs on Soil Health: A Review. Asian Journal of Plant Sciences 16(3):109-131.

6. Carmichael WW (2001) Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”. Human and Ecological Risk Assessment: An International Journal 7(5):1393-1407.

7. 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

8. Zahran HH (1999) Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Conditions and in an Arid Climate. Microbiology and Molecular Biology Reviews 63(4):968-989.

9. Birhane E, Kuyper TW, Sterck FJ, Gebrehiwot K, & Bongers F (2015) Arbuscular mycorrhiza and water and nutrient supply differently impact seedling performance of dry woodland species with different acquisition strategies. Plant Ecology & Diversity 8(3):387-399.

10. Taylor TN, Remy W, Hass H, & Kerp H (1995) Fossil Arbuscular Mycorrhizae from the Early Devonian. Mycologia 87(4):560-573.

11. Buddrus-Schiemann K, Schmid M, Schreiner K, Welzl G, & Hartmann A (2010) Root Colonization by Pseudomonas sp. DSMZ 13134 and Impact on the Indigenous Rhizosphere Bacterial Community of Barley. Microbial Ecology 60(2):381-393.

12. Bergey DH, Krieg NR, & Holt JG (1984) Bergey's manual of systematic bacteriology (Williams & Wilkins, Baltimore, MD).

13. Sah S, Singh N, & Singh R (2017) Iron acquisition in maize (Zea mays L.) using Pseudomonas siderophore. 3 Biotech 7(2):121.

14. Sharma SB, Sayyed RZ, Trivedi MH, & Gobi TA (2013) Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2:587.

15. Ruffner B, et al. (2013) Oral insecticidal activity of plant-associated pseudomonads. Environmental microbiology 15(3):751-763.

16. Jousset A, et al. (2009) Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters. The ISME journal 3(6):666-674.

17. 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.

18. 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.