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− | <p style="font-size:medium">We examined how three species of free-living nitrogen-fixing bacteria respond to the presence of the flavonoid naringenin. The three species, <i>Azorhizobium caulinodans</i> | + | <p style="font-size:medium">We examined how three species of free-living nitrogen-fixing bacteria respond to the presence of the flavonoid naringenin. The three species, <i>Azorhizobium caulinodans</i> strain ORS571, <i>Azospirillum brasilense</i> strain SP245, and <i>Herbaspirillum seropedicae</i> strain Z67, were selected because they all have potential to form different types of interactions with plant roots. <i>A. caulinodans</i> has been shown to fix nitrogen both as a free-living microbe and when in symbiosis with the semi-aquatic leguminous tree <i>Sesbania rostrata</i> [1]. <i>H. seropedicae</i> is a root endophyte and has shown potential to colonise popular crops such as wheat and maize [2]. </p> |
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− | <p style="font-size:medium">Before commencing chemotaxis studies, we needed to understand the growth characteristics of the three free-living nitrogen-fixing bacteria to be used in our project. We first examined the colony morphology of these three species in the absence of any chemoattractants. Familiarisation with the bacteria allows identification of abnormal behaviour and contamination. For colony morphology, the size after a minimum of 24 hours and morphology (shape and pigmentation) | + | <p style="font-size:medium">Before commencing chemotaxis studies, we needed to understand the growth characteristics of the three free-living nitrogen-fixing bacteria to be used in our project. We first examined the colony morphology of these three species in the absence of any chemoattractants. Familiarisation with the bacteria allows identification of abnormal behaviour and contamination. For colony morphology, the size after a minimum of 24 hours and morphology (shape and pigmentation) were recorded (Table 1, Figure 1). </p> |
<font size="2">Table 1: Qualitative analysis of <i>Azorhizobium caulinodans</i>, <i>Azospirillum brasilense</i>, <i>Herbaspirillum seropedicae</i> colonies grown on solid media.</font> | <font size="2">Table 1: Qualitative analysis of <i>Azorhizobium caulinodans</i>, <i>Azospirillum brasilense</i>, <i>Herbaspirillum seropedicae</i> colonies grown on solid media.</font> | ||
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<td>White</td> | <td>White</td> | ||
<td>Regular form, Typically raised, Entire margin</td> | <td>Regular form, Typically raised, Entire margin</td> | ||
− | <td>Colonies rarely grow to a measurable size when grown at | + | <td>Colonies rarely grow to a measurable size when grown at 30˚C on Yeast Extract Broth agar after 24 hours</td> |
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<p style="font-size:medium"><i>A. caulinodans</i> (Figure 1a): Colonies do not grow to a measurable size within 24 hours at 30 ˚C on Yeast Extract Broth agar. Colonies contain white pigmentation and are raised in elevation with an entire margin – a continuous, uninterrupted border of the colony. Colonies rarely grow larger than 2 mm whilst smaller colonies, which are much more numerous, could not be accurately measured. </p> | <p style="font-size:medium"><i>A. caulinodans</i> (Figure 1a): Colonies do not grow to a measurable size within 24 hours at 30 ˚C on Yeast Extract Broth agar. Colonies contain white pigmentation and are raised in elevation with an entire margin – a continuous, uninterrupted border of the colony. Colonies rarely grow larger than 2 mm whilst smaller colonies, which are much more numerous, could not be accurately measured. </p> | ||
− | <p style="font-size:medium"><i>A. brasilense</i> (Figure 1b): Colonies are distinguishable by their distinctive orange/pink pigmentation though both immature and dead colonies lack this pigmentation. Older colonies became ingrained into the agar, making them hard to remove without damaging the agar. Older colonies also began to wrinkle with time. The average diameter for a colony of this species after 24 hours incubation at 37 ˚C on LB | + | <p style="font-size:medium"><i>A. brasilense</i> (Figure 1b): Colonies are distinguishable by their distinctive orange/pink pigmentation, though both immature and dead colonies lack this pigmentation. Older colonies became ingrained into the agar, making them hard to remove without damaging the agar. Older colonies also began to wrinkle with time. The average diameter for a colony of this species after 24 hours incubation at 37 ˚C on LB agar was 3 mm, making <i>A. brasilense</i> the fastest growing of our nitrogen-fixing bacteria. Young <i>A. brasilense</i> colonies were shiny, round and with entire margins. These young colonies may have some pigmentation near the centre as the colony matures. This is in contrast to older colonies which maintain a different phenotype; losing their shine and gaining the odd wrinkle. Wrinkling often leads to the loss of the round shape. </p> |
<p style="font-size:medium"><i>H. seropedicae</i> (Figure 1c): the colonies take different forms depending on how the plate is inoculated. If the plate is stab-inoculated, the colony takes a rhizoid appearance (Figure 1a). If the culture is spread across the plate, then it typically takes a circular or irregular form (Figure 1b). Colonies possess a green-cream pigmentation and are raised from the surface. Most colonies were shiny and typically 1.5 mm in diameter after 24 hours at 30 ˚C. </p> | <p style="font-size:medium"><i>H. seropedicae</i> (Figure 1c): the colonies take different forms depending on how the plate is inoculated. If the plate is stab-inoculated, the colony takes a rhizoid appearance (Figure 1a). If the culture is spread across the plate, then it typically takes a circular or irregular form (Figure 1b). Colonies possess a green-cream pigmentation and are raised from the surface. Most colonies were shiny and typically 1.5 mm in diameter after 24 hours at 30 ˚C. </p> | ||
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<img src="https://static.igem.org/mediawiki/2018/b/be/T--Newcastle--AllPreservePlatesNew.png"> | <img src="https://static.igem.org/mediawiki/2018/b/be/T--Newcastle--AllPreservePlatesNew.png"> | ||
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− | <font size="2">Figure 1: Observations of bacterial preservation plates. a) <i>A. caulinodans</i> colonies grown on 1% | + | <font size="2">Figure 1: Observations of bacterial preservation plates. a) <i>A. caulinodans</i> colonies grown on 1% Yeast Extract Broth agar after incubation at 30 °C for 56 hours. Plates were inoculated via streaking. b) <i>A. brasilense</i> colonies grown on 1 % LB after incubation at 37 °C for 16 hours. Plate inoculated via streaking. c) <i>H. seropedicae</i> colonies showing circular growth on 1 % LB agar after incubation at 30 °C for 24 hours. Plates were inoculated via streaking. d) <i>H. seropedicae</i> colonies showing rhizoid growth on 1 % LB agar after incubation at 30 °C for 24 hours. Plates were stab-innoculated. </font> |
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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 <i>E. coli</i> 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. <i>H. seropedicae</i> 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"> | ||
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− | <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> | + | <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 that cell death may lead to results similar to 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"> | ||
− | <font size="2">Figure 2: Optical density at | + | <font size="2">Figure 2: Optical density at 600 nm wavelength of 4 bacterial species (<i>A. brasilense</i>, <i>A. caulinodans</i>, <i>H. seropedicae</i>, and <i>E. coli</i>) after 24 hours of growth when grown in liquid media (LB) containing different concentrations of naringenin. </font> |
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<p style="font-size:medium">To characterise chemotactic behaviour in response to naringenin, a quantitative approach is desirable. This allows for direct comparison of the strength of the response between different species. Results from a quantitative assay would also be better suited for our <a href="https://2018.igem.org/Team:Newcastle/Modelling/Community" class="black">community model</a> as it allows a ranking of bacterial responses to naringenin. </p> | <p style="font-size:medium">To characterise chemotactic behaviour in response to naringenin, a quantitative approach is desirable. This allows for direct comparison of the strength of the response between different species. Results from a quantitative assay would also be better suited for our <a href="https://2018.igem.org/Team:Newcastle/Modelling/Community" class="black">community model</a> as it allows a ranking of bacterial responses to naringenin. </p> | ||
− | <font size="2">Table 2: Colony forming units of four bacterial species from capillaries containing 1 µl 100 µM naringenin or motility buffer solution (10 mM potassium phosphate, 0.1 mM EDTA, 10 mM glucose, pH 7.0) after 60 minutes open-end submersion in sterile conditions at room temperature/pressure. Values are mean cfu.μl<sup>-1</sup>. Difference between colony counts from capillaries containing naringenin or motility buffer was non-significant for all species (P>0.05). </font> | + | <font size="2">Table 2: Colony forming units of four bacterial species from capillaries containing 1 µl 100 µM naringenin or motility buffer solution (10 mM potassium phosphate, 0.1 mM EDTA, 10 mM glucose, pH 7.0) after 60 minutes open-end submersion in sterile conditions at room temperature/pressure. Values are mean cfu.μl<sup>-1</sup>. Difference between colony counts from capillaries containing naringenin or motility buffer was non-significant for all species (P > 0.05). </font> |
<table id="protocols"> | <table id="protocols"> | ||
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<img src="https://static.igem.org/mediawiki/2018/1/1e/T--Newcastle--HerbaspirillumseropedicaeCapillaryPlates.png"> | <img src="https://static.igem.org/mediawiki/2018/1/1e/T--Newcastle--HerbaspirillumseropedicaeCapillaryPlates.png"> | ||
<p> </p> | <p> </p> | ||
− | <font size="2">Figure 3: a) Growth of <i>H. seropedicae</i> on Typtone and Yeast Extract agar inoculated with contents of a 1 µl capillary containing 100 µM naringenin after 60 minutes open-end submersion in bacterial solution. Plate was incubated for 24 hours at 30 °C. b) Growth of <i>H. seropedicae</i> on 1 % LB agar inoculated with contents of a 1 µl capillary containing motility buffer after 60 minutes open-end submersion in bacterial solution. Plates were | + | <font size="2">Figure 3: a) Growth of <i>H. seropedicae</i> on Typtone and Yeast Extract agar inoculated with contents of a 1 µl capillary containing 100 µM naringenin after 60 minutes open-end submersion in bacterial solution. Plate was incubated for 24 hours at 30 °C. b) Growth of <i>H. seropedicae</i> on 1 % LB agar inoculated with contents of a 1 µl capillary containing motility buffer after 60 minutes open-end submersion in bacterial solution. Plates were inoculated via streaking technique and incubated for 24 hours at 30 °C. |
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<p style="font-size:medium"><b><u>Method 1:</u></b></p> | <p style="font-size:medium"><b><u>Method 1:</u></b></p> | ||
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− | <p style="font-size:medium">The original method utilised 0.75 % LB agar plates with | + | <p style="font-size:medium">The original method utilised 0.75 % LB agar plates with 10 μl of 200 μM naringenin applied to one side of the plate and a sterile water control to the other. Bacteria were inoculated into the centre of the plate and it was hypothesised that colony growth would be distorted towards the side that contained naringenin, in the case of positive chemotactic behaviour. This assay was conducted with <i>A. brasilense</i> and <i>E. coli</i>. Neither bacterium showed a growth response favouring either the side with naringenin or the control. </p> |
<p style="font-size:medium">From the results in this iteration, several key elements were identified that were incorporated moving forward. For example, as bacterial growth exhibits inherent variability, a qualitative assay may not be sufficient to identify differences in behaviour in response to different treatments. As such, a more qualitative approach was adopted for future assays. Another issue noted was that there is the potential that the agar percentage was too high resulting in poor diffusion through the medium. This may also have impacted the bacteria’s ability to move and grow towards the naringenin source.</p> | <p style="font-size:medium">From the results in this iteration, several key elements were identified that were incorporated moving forward. For example, as bacterial growth exhibits inherent variability, a qualitative assay may not be sufficient to identify differences in behaviour in response to different treatments. As such, a more qualitative approach was adopted for future assays. Another issue noted was that there is the potential that the agar percentage was too high resulting in poor diffusion through the medium. This may also have impacted the bacteria’s ability to move and grow towards the naringenin source.</p> | ||
<p> </p> | <p> </p> | ||
<p style="font-size:medium"><b><u>Method 2:</u></b></p> | <p style="font-size:medium"><b><u>Method 2:</u></b></p> | ||
<p> </p> | <p> </p> | ||
− | <p style="font-size:medium">The second iteration of agar assays reduced the agar concentration to 0.5 % and the naringenin concentration to | + | <p style="font-size:medium">The second iteration of agar assays reduced the agar concentration to 0.5 % and the naringenin concentration to 100 μM to align with the findings of the impact of naringenin on growth rate. The plate was also laid out in a more quantifiable manner. This followed concerns of the chemoattractant diffusing onto the side of the control when on the same plate. In this method, the distance of bacterial growth towards the naringenin/control source was measured (Table 5). </p> |
<font size="2">Table 5: Mean distance of colony growth towards either naringenin or control source of <i>A. brasilense</i>, <i>A. caulinodans</i>, <i>H. seropedicae</i> and <i>E. coli</i> measured from the point of inoculation after 24 hours incubation. Distance is given in mm</font> </div> | <font size="2">Table 5: Mean distance of colony growth towards either naringenin or control source of <i>A. brasilense</i>, <i>A. caulinodans</i>, <i>H. seropedicae</i> and <i>E. coli</i> measured from the point of inoculation after 24 hours incubation. Distance is given in mm</font> </div> | ||
<table id="protocols"> | <table id="protocols"> | ||
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<p style="font-size:medium">The third and final iteration of agar assays was based on the gradient plate experiment used by Reyes-Darias et al. (2016) [9]. In this variant, 0.25 % Minimal A Salt agar was utilised and the naringenin concentration was further reduced to 50 μM. The concentration gradients were also left for 16 hours at 4 ˚C in order to form instead of 12 hours at room temperature. Initially, bacterial species were inoculated at different distances from the centre line where the naringenin or control was added; this interval increased by 5 mm until 40 mm. After analysing initial results, the inoculation distance was changed to reflect that which gave the best response index. The control was also altered to 1.5 % (v/v) ethanol as the method of dissolving naringenin was changed to be within the same percentage.</p> | <p style="font-size:medium">The third and final iteration of agar assays was based on the gradient plate experiment used by Reyes-Darias et al. (2016) [9]. In this variant, 0.25 % Minimal A Salt agar was utilised and the naringenin concentration was further reduced to 50 μM. The concentration gradients were also left for 16 hours at 4 ˚C in order to form instead of 12 hours at room temperature. Initially, bacterial species were inoculated at different distances from the centre line where the naringenin or control was added; this interval increased by 5 mm until 40 mm. After analysing initial results, the inoculation distance was changed to reflect that which gave the best response index. The control was also altered to 1.5 % (v/v) ethanol as the method of dissolving naringenin was changed to be within the same percentage.</p> | ||
− | <p style="font-size:medium">The response index, developed by Pham and Parkinson [4], accounts for a ratio between the edge of the colony nearest the chemoattractant source and the edge furthest from the same source. This ratio is then used to determine if there has been positive chemotaxis (RI >0.52), no effect (RI = 0.48-0.52) or negative chemotaxis (RI <0.48).</p> | + | <p style="font-size:medium">The response index, developed by Pham and Parkinson [4], accounts for a ratio between the edge of the colony nearest the chemoattractant source and the edge furthest from the same source. This ratio is then used to determine if there has been positive chemotaxis (RI > 0.52), no effect (RI = 0.48-0.52) or negative chemotaxis (RI < 0.48).</p> |
− | <p style="font-size:medium">Results (Table 6) indicated that both <i>A. brasilense</i> and <i>H. seropedicae</i> experienced positive chemotaxis towards 50 μM between distances of 5-25 mm and 5-10 mm respectively. As such, further investigation utilised the distance that corresponded with the greatest RI value ( | + | <p style="font-size:medium">Results (Table 6) indicated that both <i>A. brasilense</i> and <i>H. seropedicae</i> experienced positive chemotaxis towards 50 μM between distances of 5-25 mm and 5-10 mm respectively. As such, further investigation utilised the distance that corresponded with the greatest RI value (15 mm and 10 mm respectively). For <i>H. seropedicae</i>, the colonies nearer the centre line again showed more constricted halos which may indicate that the naringenin concentration may still be too high. The response index of the control for all species at 5 mm was < 0.48, suggesting chemorepulsion. This was anticipated as the control contains ethanol which possesses known antimicrobial properties and is commonly used to disinfect lab equipment.</p> |
− | <font size="2">Table 6: Average Response Index and standard error of <i>A. caulinodans</i>, <i>A. brasilense</i>, <i>H. seropedicae</i> and <i>E. coli</i> colonies grown on 0.25% Minimal A Salt agar containing a gradient of either | + | <font size="2">Table 6: Average Response Index and standard error of <i>A. caulinodans</i>, <i>A. brasilense</i>, <i>H. seropedicae</i> and <i>E. coli</i> colonies grown on 0.25 % Minimal A Salt agar containing a gradient of either 100 µM naringenin or 1.5 % ethanol (control). RI = D1/(D1+D2) in which D1 represents distance between colony edge nearest chemical source to site of inoculation whilst D2 represents distance between colony edge furthest from chemical source to site of innoculation [4]. Bacteria were innoculated 15mm (<i>A. brasilense</i> and <i>E. coli</i>) or 10 mm (<i>A. caulinodans</i> and (<i>H. seropedicae</i>) from naringenin source and incubated at 30 ˚C.</font> |
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− | <p style="font-size:medium">The response index for <i>E. coli</i> and <i>A. caulinodans</i> indicated negative chemotaxis in response to naringenin. This may be due to the fact that the naringenin wass dissolved in 1.5% ethanol which is commonly used to to sterilise due to ethanol's antimicrobial properties. As the other two species of nitrogen-fixers were demonstrated to show chemoattraction and both of which were motile, unlike <i>A. caulinodans</i>, it may be possible that the loss of motility combined with the antimicrobial properties of ethanol are triggering this result. This would mean the experimental set-up was not appropriate and thus requires further work. This will be work for the future.</p> | + | <p style="font-size:medium">The response index for <i>E. coli</i> and <i>A. caulinodans</i> indicated negative chemotaxis in response to naringenin. This may be due to the fact that the naringenin wass dissolved in 1.5 % ethanol which is commonly used to to sterilise due to ethanol's antimicrobial properties. As the other two species of nitrogen-fixers were demonstrated to show chemoattraction and both of which were motile, unlike <i>A. caulinodans</i>, it may be possible that the loss of motility combined with the antimicrobial properties of ethanol are triggering this result. This would mean the experimental set-up was not appropriate and thus requires further work. This will be work for the future.</p> |
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Revision as of 23:53, 16 October 2018