Difference between revisions of "Team:Newcastle/InterLab"

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                 <p> 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:</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 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:</font></p>
  
 
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                 <p>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, plasmid backbone and protocol workflow are shown in Figure 2. </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, plasmid backbone and protocol workflow are shown in Figure 2.</font></p>
  
 
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<p><font size="3"><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. </font></center></p>
 
<p><font size="3"><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. </font></center></p>
  
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  <p>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 <a href="https://2018.igem.org/Team:Newcastle/Measurement">measurements page</a>.</p>
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  <p><font size="3”>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 <a href="https://2018.igem.org/Team:Newcastle/Measurement">measurements page</a>.</font></p>
  
 
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Revision as of 12:58, 16 October 2018

Alternative Roots

InterLab Study

Calibrations Cell Measurement Results
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Calibration

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.



  • 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 1 A).
  • 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.

    InterLab Protocol

    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. C: Interlab study protocol workflow.

    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.

    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.





    InterLab

    REFERENCES

    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

    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

    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.

    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

    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

    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).

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

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

    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.

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

    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

    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

    13. Bergey, D. H., et al. (1984). Bergey's manual of systematic bacteriology. Baltimore, MD, Williams & Wilkins.

    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.

    15. Gross, H. and J. Loper (2009). Genomics of secondary metabolite production by Pseudomonas spp.

    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.

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

    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

    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.

    20. Maheshwari DK (2012) Bacteria in Agrobiology: Plant Probiotics (Springer Berlin Heidelberg).

    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.

    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].