Difference between revisions of "Team:Newcastle/InterLab"

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                 <h1 class="display-2">2018 Interlab study aims</h1>
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                <p>A weakness in the measurement of fluorescence relative to optical density (OD), 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><font size="3">Alternative Roots participated in the 2018 InterLab study. Three calibration steps were carried out prior to any experimental measurements being taken. Absorbance and fluorescence values were measured in 96-well plates using a Thermofisher Varioskan Lux plate reader (Thermofisher Scientific). Absorbance was measured at 600 nm and converted to a comparable OD<sub>600</sub>. Fluorescence was measured at 525 nm with excitation at 485 nm with a 12 nm bandpass width. All readings took place at 25 °C and pathlength correction was disabled. The calibrations were:</p>
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<p><font size="3">1. A LUDOX CL-X 45 % colloidal silica suspension was used to calculate a conversion factor for the Abs<sub>600</sub> value measured by the plate reader to a comparable OD<sub>600</sub> value, considering path length and well volume (Table 1). Abs<sub>600</sub> of 1:2 dilutions of LUDOX silica suspension were taken in triplicate and a reference OD<sub>600</sub> of 0.063 (the reference value for 100 µL of LUDOX CL-X in a well of a standard 96-well flat-bottom black with clear bottom plate) divided by the mean measured value to give a conversion factor.  </font></p>
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<p><font size="2"><center>Table 1. Optical density readings for LUDOX CL-X 45% colloidal silica suspension and water used to calculate the conversion factor for absorbance readings to OD<sub>600</sub> readings for plate reader measurements.
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<p><font size="3">2. A standard curve was prepared by measuring the OD<sub>600</sub> of serial dilutions of monodisperse silica microspheres, with similar light scattering properties to <i>E. coli</i> cells. This was used to standardise OD readings across labs (Figure 1A). </p>
  
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<p><font size="3">3. A fluorescence standard curve was created by measuring the fluorescence of serial dilutions of the small molecule fluorescein. This  has similar excitation and emission characteristics to GFP allowing conversion of fluorescence readings to an equivalent fluorescein concentration. Calibrations allowed expression measurement in units of fluorescence per OD and molecules of equivalent fluorescein (MEFL) per cell (Figure 1B). </p>
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<p><font size="2"><center>Figure 1. Curves used to calibrate A; Fluorescein per OD using dilutions of fluorescein and B; molecules of equivalent fluorescein per particle using dilutions of solutions of monodisperse silica microspheres.</font></center></p>
  
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                 <h1 class="display-2">Problems With Fertiliser</h1>
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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><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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                <p><font size="3">Following these calibration steps, two transformed colonies for each test device and both control plasmids were used to inoculate lysogeny broth (LB) medium containing 25 µg/L of chloramphenicol (CAM) and incubated overnight at 37 °C with shaking at 220 rpm. Overnight cultures were diluted 1:10 and the OD<sub>600</sub> adjusted to 0.02 with LB with CAM to a final volume of 12 mL. Fluorescence and Abs<sub>600</sub> were taken at 0h and 6 hours of incubation at 37 °C with 220 rpm shaking. Test devices and plasmid backbone are shown in Figure 2.</font></p>
  
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<p><font size="2"><center>Figure 2. A) test devices transform into DH5α for the iGEM InterLab study. A negative control device consisting of a tetracycline resistance gene (BBa_R0040) with no associated promoter, RBS or terminator sequences was also transformed into DH5α. B) All test devices were transformed into DH5α using the pSB1C3 plasmid backbone with chloramphenicol for selection (adapted from LabGenius plasmid viewer).</font></center></p>
  
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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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<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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                <h1 class="display-2"><font color="white">Advantages of Contained Agriculture</font></h1>
 
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                <p><font size="3">Results for OD<sub>600</sub> and fluorescence measurements after 6 hours are shown in Figure 3.</font></p>
  
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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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<li>Reducing the Carbon footprint of crop production due to reduced food millage.</li>
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<li>No agricultural run-off.</li>
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<li>Limited need for pesticides and herbicides.</li>
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<li>Safer crops as there is less risk of contamination.</li>
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<li>Reduced spoilage because of shorter transportation times and reduced handling.</li>
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<li>Less agricultural pollution.</li></ul>
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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><font size="2"><center>Figure 3. OD<sub>600</sub> (A) and fluorescence (B) InterLab study results for positive and negative controls and all test devices after 6 hours. Tests were performed in duplicate using two colonies for each test device, results for which are shown separately to account for colony-to-colony variation. </center></font></p>
  
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<p><font size="3">Results for both 0 and 6 hours were converted into fluorescence per OD<sub>600</sub> and fluorescence per cell using the previously described calibrations (Figure 4). While the InterLab study is an opportunity for crowdsourcing a large amount of data on the inherent variability of a given biological system, it does not account for variations within data sets. Our team attempted to seek out and address such sources of variation, investigating biodesign automation of protocols, use of internal standards and effects of media composition. Further detail and results are available on the <a href="https://2018.igem.org/Team:Newcastle/Measurement"class="black">measurements page</a>.</font></p>
  
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<p><font size="2"><center>Figure 4. iGEM InterLab study results for positive and negative controls and all test devices. Fluorescence was measured at 525 nm and readings were taken at 0 hours and 6 hours in a 96 well plate.  A: Fluorescence per OD<sub>600</sub>, B: Fluorescence per <i>Escherichia coli</i> cell, calibrated to a standard curve of LUDOX silica beads. </center></font></p>
  
 
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<p class="about-para"><strong>Attributions: Matthew Burridge, Kyle Stanforth, Sam Went
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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">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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Latest revision as of 02:04, 18 October 2018

Alternative Roots

InterLab Study

Calibrations Protocol Results

Calibration

Alternative Roots participated in the 2018 InterLab study. Three calibration steps were carried out prior to any experimental measurements being taken. Absorbance and fluorescence values were measured in 96-well plates using a Thermofisher Varioskan Lux plate reader (Thermofisher Scientific). Absorbance was measured at 600 nm and converted to a comparable OD600. Fluorescence was measured at 525 nm with excitation at 485 nm with a 12 nm bandpass width. All readings took place at 25 °C and pathlength correction was disabled. The calibrations were:

1. A LUDOX CL-X 45 % colloidal silica suspension was used to calculate a conversion factor for the Abs600 value measured by the plate reader to a comparable OD600 value, considering path length and well volume (Table 1). Abs600 of 1:2 dilutions of LUDOX silica suspension were taken in triplicate and a reference OD600 of 0.063 (the reference value for 100 µL of LUDOX CL-X in a well of a standard 96-well flat-bottom black with clear bottom plate) divided by the mean measured value to give a conversion factor.

Table 1. Optical density readings for LUDOX CL-X 45% colloidal silica suspension and water used to calculate the conversion factor for absorbance readings to OD600 readings for plate reader measurements.



2. A standard curve was prepared by measuring the OD600 of serial dilutions of monodisperse silica microspheres, with similar light scattering properties to E. coli cells. This was used to standardise OD readings across labs (Figure 1A).

3. A fluorescence standard curve was created by measuring the fluorescence of serial dilutions of the small molecule fluorescein. This has similar excitation and emission characteristics to GFP allowing conversion of fluorescence readings to an equivalent fluorescein concentration. Calibrations allowed expression measurement in units of fluorescence per OD and molecules of equivalent fluorescein (MEFL) per cell (Figure 1B).



Figure 1. Curves used to calibrate A; Fluorescein per OD using dilutions of fluorescein and B; molecules of equivalent fluorescein per particle using dilutions of solutions of monodisperse silica microspheres.

Protocol

Following these calibration steps, two transformed colonies for each test device and both control plasmids were used to inoculate lysogeny broth (LB) medium containing 25 µg/L of chloramphenicol (CAM) and incubated overnight at 37 °C with shaking at 220 rpm. Overnight cultures were diluted 1:10 and the OD600 adjusted to 0.02 with LB with CAM to a final volume of 12 mL. Fluorescence and Abs600 were taken at 0h and 6 hours of incubation at 37 °C with 220 rpm shaking. Test devices and plasmid backbone are shown in Figure 2.

Figure 2. A) test devices transform into DH5α for the iGEM InterLab study. A negative control device consisting of a tetracycline resistance gene (BBa_R0040) with no associated promoter, RBS or terminator sequences was also transformed into DH5α. B) All test devices were transformed into DH5α using the pSB1C3 plasmid backbone with chloramphenicol for selection (adapted from LabGenius plasmid viewer).

Results

Results for OD600 and fluorescence measurements after 6 hours are shown in Figure 3.

Figure 3. OD600 (A) and fluorescence (B) InterLab study results for positive and negative controls and all test devices after 6 hours. Tests were performed in duplicate using two colonies for each test device, results for which are shown separately to account for colony-to-colony variation.

Results for both 0 and 6 hours were converted into fluorescence per OD600 and fluorescence per cell using the previously described calibrations (Figure 4). While the InterLab study is an opportunity for crowdsourcing a large amount of data on the inherent variability of a given biological system, it does not account for variations within data sets. Our team attempted to seek out and address such sources of variation, investigating biodesign automation of protocols, use of internal standards and effects of media composition. Further detail and results are available on the measurements page.

Figure 4. iGEM InterLab study results for positive and negative controls and all test devices. Fluorescence was measured at 525 nm and readings were taken at 0 hours and 6 hours in a 96 well plate. A: Fluorescence per OD600, B: Fluorescence per Escherichia coli cell, calibrated to a standard curve of LUDOX silica beads.





References & Attributions

Attributions: Matthew Burridge, Kyle Stanforth, Sam Went