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− | <p style="font-size:medium">From initial iterations of our <a href="https://2018.igem.org/Team:Newcastle/Modelling/Community" class="black">community model</a>, it became apparent that quantitative data on the growth rates of the bacteria were required in order to inform the model. For this, we observed changes in absorbance at 600 nm over 72 hours of the three nitrogen-fixing bacteria and E. coli in liquid culture at 30 °C using a ThermoFisher Scientific Varioskan LUX Microplate Reader.</p> | + | <p style="font-size:medium">From initial iterations of our <a href="https://2018.igem.org/Team:Newcastle/Modelling/Community" class="black">community model</a>, it became apparent that quantitative data on the growth rates of the bacteria were required in order to inform the model. For this, we observed changes in absorbance at 600 nm over 72 hours of the three nitrogen-fixing bacteria and <i>E. coli</i> in liquid culture at 30 °C using a ThermoFisher Scientific Varioskan LUX Microplate Reader.</p> |
<p style="font-size:medium">The data showed that <i>A. brasilense</i> grew at a slow, steady rate before sharply dying off after approximately 60 hours. The slow growth rate is likely to be because its optimal growth temperature is 37 °C rather than 30 °C. H. seropedicae and <i>A. caulinodans</i> showed very similar growth curves when grown at 30 °C: initial growth rate was very fast and then growth became very slow or static after 20 hours. <i>E. coli</i> grew at a medium pace to begin with and steadily slowed down with time. </p> | <p style="font-size:medium">The data showed that <i>A. brasilense</i> grew at a slow, steady rate before sharply dying off after approximately 60 hours. The slow growth rate is likely to be because its optimal growth temperature is 37 °C rather than 30 °C. H. seropedicae and <i>A. caulinodans</i> showed very similar growth curves when grown at 30 °C: initial growth rate was very fast and then growth became very slow or static after 20 hours. <i>E. coli</i> grew at a medium pace to begin with and steadily slowed down with time. </p> | ||
<img src="https://static.igem.org/mediawiki/2018/0/0c/T--Newcastle--ChemotaxisGrowthCurveGraph.png"> | <img src="https://static.igem.org/mediawiki/2018/0/0c/T--Newcastle--ChemotaxisGrowthCurveGraph.png"> | ||
<p> </p> | <p> </p> | ||
− | <font size="2">Figure 2: Growth curves showing changes in absorbance at 600 nm of <i>E. coli, A. | + | <font size="2">Figure 2: Growth curves showing changes in absorbance at 600 nm of <i>E. coli, A. caulinodans, H. seropedicae,</i> and <i> A. brasilense</i> in LB at 30 °C for 72 hours.</font> |
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− | <p style="font-size:medium">Initial research for the Alternative Roots project noted that naringenin possesses antimicrobial properties, particularly | + | <p style="font-size:medium">Initial research for the Alternative Roots project noted that naringenin possesses antimicrobial properties, particularly towards <i>E. coli</i> [3]. As <i>E. coli</i> (DH5α) was to be used as both a control in our chemotaxis assays and as the organism in which our naringenin biosynthesis operon would first be assembled, it was deemed important to characterise the effect of increasing naringenin concentrations on growth rates of both our free-living nitrogen-fixing bacteria, and <i>E. coli</i> in LB medium. This was essential to guide the chemotaxis assays enabling an understanding of naringenin concentrations which would not have detrimental impacts upon the cell. If cell health is impaired, then there is potential for cell death to lead to the appearance of chemorepulsion. This is particularly problematic when applying the response index as a semi-quantitative measure of chemotactic response as the method utilises ratios between colony edges to determine the significance of chemotaxis [4].</p> |
<img src="https://static.igem.org/mediawiki/2018/9/99/T--Newcastle--ChemotaxisNaringeninKillCurve2.png"> | <img src="https://static.igem.org/mediawiki/2018/9/99/T--Newcastle--ChemotaxisNaringeninKillCurve2.png"> | ||
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<p> </p> | <p> </p> | ||
+ | <p style="font-size:medium">While completing microscopy evaluations, we utilised the ibidi µ-Slide III 3-in-1 Chemotaxis Microscopy Slide. This specialised microscopy slide was designed to allow real time observations of chemotactic behaviour in response to an adjustable chemical gradient. Through working with experts at ibidi, we were able to successfully seed all 4 species of our bacteria onto the uncoated, hydrophobic variant of the slide. This would allow us to begin quantifying naringenin chemotaxis in a modern and easily repeatable manner. Continued work with ibidi in the future demonstrates the exciting potential to quantify naringenin to further improve the design of our community model in the future. More information on ibidi and the µ-Slide III 3-in-1 Chemotaxis Microscopy Slide can be found <a href="https://ibidi.com/channel-slides/55--slide-iii-3in1.html" class="black"> | ||
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<td><i>H. seropedicae</i> (Z67)</td> | <td><i>H. seropedicae</i> (Z67)</td> | ||
− | <td> | + | <td>0.625</td> |
− | <td> | + | <td>0.5</td> |
+ | <td>Positive</td> | ||
</tr> | </tr> | ||
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</section> | </section> | ||
− | <section id=' | + | <section id='Conclusions' class="s-services"> |
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Revision as of 21:34, 16 October 2018