Latest revision as of 15:34, 10 November 2018

Alternative Roots

Exploring Bacterial Chemotaxis

Introduction Colony Morphology Growth in LB Naringenin & Growth Quantification Microscopy Chemotaxis on Agar Conclusions

Naringenin Chemotaxis

An Introduction

We examined how three species of free-living nitrogen-fixing bacteria respond to the presence of the flavonoid naringenin. The three species, Azorhizobium caulinodans  strain ORS571,  Azospirillum brasilense  strain SP245, and  Herbaspirillum seropedicae  strain Z67, were selected because they all have potential to form different types of interactions with plant roots. A. caulinodans  has been shown to fix nitrogen both as a free-living microbe and when in symbiosis with the semi-aquatic leguminous tree Sesbania rostrata (1).  H. seropedicae  is a root endophyte and has shown potential to colonise popular crops such as wheat and maize (2).

Bacteria Characterisation

Colony Morphology

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).

Table 1. Qualitative analysis of Azorhizobium caulinodans, Azospirillum brasilense, and Herbaspirillum seropedicae colonies grown on solid media.

Species (Strain)	Colony Pigmentation	Colony Morphology	Points of Interest
Azorhizobium caulinodans (ORS571)	White	Regular form, Typically raised, Entire margin	Colonies rarely grow to a measurable size when grown at 30 ˚C on Yeast Extract Broth agar after 24 hours
Azospirillum brasilense (SP245)	Orange/Pink	Non-slimy, Regular and round form, Entire margins	Both immature and dead colonies lack the orange/pink pigment, Colonies wrinkle with age
Herbaspirillum seropedicae (Z67)	Cream/Light Green	Circular or Irregular form (occasionally rhizoid), Raised elevation, Shiny	Colonies took on a different morphology depending on how the media was inoculated; stab-inoculation lead to rhizoid form while spreading leads to circular/irregular form

A. caulinodans (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.

A. brasilense (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 A. brasilense the fastest growing of our nitrogen-fixing bacteria. Young A. brasilense 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.

H. seropedicae (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 1d). If the culture is spread across the plate, then it typically takes a circular or irregular form (Figure 1c). 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.

Figure 1. Observations of bacterial preservation plates. a) A. caulinodans colonies grown on 1 % Yeast Extract Broth agar after incubation at 30 °C for 56 hours. Plates were inoculated via streaking. b) A. brasilense colonies grown on 1 % LB after incubation at 37 °C for 16 hours. Plate inoculated via streaking. c) H. seropedicae colonies showing circular growth on 1 % LB agar after incubation at 30 °C for 24 hours. Plates were inoculated via streaking. d) H. seropedicae colonies showing rhizoid growth on 1 % LB agar after incubation at 30 °C for 24 hours. Plates were stab-inoculated.

Growth Rates in Liquid Media

From initial iterations of our community model, 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 (Figure 2).

The data showed that A. brasilense 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 A. caulinodans 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. E. coli grew at a medium pace to begin with and steadily slowed down with time.

Figure 2. Growth curves showing changes in absorbance at 600 nm of E. coli, A. caulinodans, H. seropedicae, and A. brasilense in LB at 30 °C for 70 hours. n=4 replicates, error bars indicate standard error of the mean.

Effect of Naringenin on Growth Rate in Liquid Culture

Initial research for the Alternative Roots project noted that naringenin possesses antimicrobial properties, particularly towards E. coli (3). As E. coli 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 E. coli 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).

Figure 3. Optical density at 600 nm wavelength of 4 bacterial species (A. brasilense, A. caulinodans, H. seropedicae, and E. coli) after 24 hours of growth when grown in liquid media (LB) containing different concentrations of naringenin. n=3 replicates, error bars indicate standard error of the mean.

All species successfully grew in the presence of 0-150 μM naringenin (Figure 3). However, it was noted that when the concentration of naringenin exceeded 100 μM, the amount of error also increases. This suggests that naringenin begins to have a greater impact on some, but not all, bacteria in the solution. As such, naringenin concentrations of <100 μM were used as part of subsequent chemotaxis assays to avoid negatively impacting bacterial growth.

Characterising Chemotactic Behaviour

Quantification Utilising Capillaries

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 community model as it allows a ranking of bacterial responses to naringenin.

Table 2: Colony forming units of four bacterial species from capillaries containing 1 µl 100 µM naringenin or chemotaxis buffer solution (10 mM potassium phosphate, 0.1 mM EDTA, 10 mM glucose, pH 7.0) after 45 minutes open-end submersion in sterile conditions at room temperature/pressure. Values are mean cfu.μl^-1. Difference between colony counts from capillaries containing naringenin or chemotaxis buffer was non-significant for all species (P > 0.05).

Species (Strain)	Colony Count (Naringenin)	± Standard Error	Colony Count (Control)	± Standard Error	Significant Difference
A. caulinodans (ORS571)	0	0	0	0	No
A. brasilense (SP245)	0	0	0	0	No
H. seropedicae (Z67)	458	86	376	236	No
E. coli (DH5α)	0	0	0	0	No

After 24 hours incubation at either 30 °C (A. caulinodans and H. seropedicae) or 37 °C (A. brasilense and E. coli), the number of colonies which grew on the LB agar plate was counted (Table 2). The results showed that of the four test bacterial species, only one was able to move into the capillary. This species was H. seropedicae which was able to move successfully into capillaries containing either the control (buffer solution) or the chemoattractant. This was demonstrated by the growth of colonies on LB agar from the contents of each capillary (Figure 4). Both methods of agar inoculation (spreading and pipetteing) lead to colony growth.

After counting colonies from the contents of both the control and naringenin capillaries, no significant difference between mean colony count of the two conditions was observed. The results therefore show no evidence for positive chemotaxis using this method. It should be considered, however, that H. seropedicae was the only species that demonstrated growth on agar, and therefore the only one able to enter the capillaries. We concluded that this methodology is not yet sufficiently optimised for our application and may be having a confounding effect upon chemotactic response.

Figure 4. a) Growth of H. seropedicae on Typtone and Yeast Extract agar inoculated with contents of a 1 µl capillary containing 100 µM naringenin after 45 minutes open-end submersion in bacterial solution. Plate was incubated for 24 hours at 30 °C. b) Growth of H. seropedicae on 1 % LB agar inoculated with contents of a 1 µl capillary containing chemotaxis buffer after 45 minutes open-end submersion in bacterial solution. Plates were inoculated via streaking technique and incubated for 24 hours at 30 °C.

Microscopy Observations

An alternative method of observing chemotactic responses is through the use of microscopy. Brightfield microscopy allows direct observations of bacterial responses. This will allow comparisons of motility and morphology from our experimental data to that of the published literature that was used to underpin our first iteration of the community model. Using microscopy enables the development of a cell density:optical density index (CD:OD index), a method of converting the two values. This index was also used in the community model to adapt the growth curve data collected during bacterial characterisation in standard laboratory conditions.

The CD:OD index was produced utilising data collected from a haemocytometer. A haemocytometer is a specialised microscopy slide of a known volume, it also contains a grid at the centre. By counting the number of cells in 16 squares at the top left and performing a series of mathematical calculations, we were able to determine cell density. By utilising a spectrophotometer, we were also able to take a reading of the optical density (600 nm) and thus link the two together (Table 3).

A. brasilense was unable to be counted accurately. This was as the bacteria was difficult to view under the microscope due to human limitations, in addition to highly variable optical density readings but consistently low cell densities despite more than sufficient incubation times. This happened throughout all replicates and as such results for this species have been omitted. Exploration of why this occurred shall be work for the future with the potential alternative approach of utilising flow cytometry.

Table 3: Cell density (cells.ml^-1) of  A. caulinodans, H. seropedicae and E. coli at different optical densities

Species (Strain)	Optical Density	Cell Density
A. caulinodans (ORS571)	1.00	3.14x10⁵
H. seropedicae (Z67)	0.05	1.05x10⁶
E. coli (DH5α)	1.00	7.4x10⁸

Utilising a haemocytometer to count cells also allows observations of cell morphology and behaviour. This was used to an advantage as it allowed us to explore whether our bacteria’s morphology aligns with the literature that was followed to produce the community model. It also enabled observations of motility which was important after theories that the species were no longer motile which is why only H. seropedicae showed movement into the capillary during the attempt to quantify chemotaxis.

Table 4: Microscopy observations of diameter (µm), cell length (µm), and motility in A. brasilense, A. caulinodans, and H. seropedicae on a haemocytometer at 40x objective compared to information in utilised literature.

Species (Strain)	Length in Literature	Mean Diameter	Cell Length in Literature	Mean Cell Length	Motile (Y/N)
A. caulinodans (ORS571)	1.5-2.5(5)	1.9	0.5-0.6 (5)	0.5	No
A. brasilense (SP245)	2.1-3.8 (6)	2.0	1.0 (6)	0.7	Yes
H. seropedicae (Z67)	1.5-5 (7)	2.4	0.7 (7)	0.7	Yes (Highly)

While completing microscopy evaluations, we utilised the ibidi µ-Slide III 3-in-1 Chemotaxis Microscopy Slide provided by our sponsors at ibidi. 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 here.

Chemotaxis on Agar

Our third approach to understanding bacterial chemotaxis involved both qualitative and semi-quantitative analysis. Growth of the nitrogen-fixing bacteria on solid media was observed to understand chemotactic behaviour in response to naringenin.

Multiple different variants of agar assays were conducted to optimise the methodology.

Method 1:

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 A. brasilense and E. coli. Neither bacterium showed a growth response favouring either the side with naringenin or the control.

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 quantitative 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.

Method 2:

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).

Table 5: Mean distance of colony growth towards either naringenin or control source of A. brasilense, A. caulinodans, H. seropedicae and E. coli measured from the point of inoculation after 24 hours incubation. Distance is given in mm.

Species (Strain)	Growth Distance Towards Naringenin	Growth Distance Towards Control
A. caulinodans (ORS571)	3.70	4.01
A. brasilense (SP245)	7.32	7.10
H. seropedicae (Z67)	5.87	5.66
E. coli (DH5α)	8.33	7.67

Once again, no evidence of chemotaxis towards naringenin was observed in any species. It was noted that the ‘halo’ around H. seropedicae colonies on plates containing naringenin were constricted and more closely situated to the colony margin. Through consulting the literature, it was revealed that high concentrations of naringenin can repress genes involved in chemotactic behaviour in this species (8). This may provide an explanation as to why chemotaxis was not observed in this species.

Method 3:

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.

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).

Results (Table 6) indicated that both A. brasilense and H. seropedicae 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 H. seropedicae, 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.

Table 6: Average Response Index and standard error of A. caulinodans, A. brasilense, H. seropedicae and E. coli colonies grown on 0.25 % Minimal A Salt agar containing a gradient of either 50 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 inoculation (4). Bacteria were inoculated 15 mm (A. brasilense and E. coli) or 10 mm (A. caulinodans and H. seropedicae) from naringenin source and incubated at 30 ˚C.

Species (Strain)	Naringenin Response Index	Control Response Index	Chemotactic Response
A. caulinodans (ORS571)	0.478	0.473	Negative
A. brasilense (SP245)	0.552	0.515	Positive
H. seropedicae (Z67)	0.625	0.5	Positive
E. coli (DH5α)	0.472	0.493	Negative

The response index for E. coli and A. caulinodans indicated negative chemotaxis in response to naringenin. This may be due to the fact that the naringenin was 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 A. caulinodans, 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.

Conclusions

After successfully characterising how A. brasilense, A. caulinodans, H. seropedicae and E. coli behave in a laboratory environment through means of understanding colonies and growth rates, we began to explore bacterial chemotaxis toward naringenin. While it may be possible to observe this behaviour in a quantitative fashion via microscopy or microfluidic methods. These methods, from the data gathered from these series of experiments, require a higher level of optimisation than semi-quantitative based methods.

Importantly, we were able to successfully demonstrate chemotaxis of A. brasilense and H. seropedicae toward 50 µM naringenin, these results came from semi-quantitative agar-based assays. While no evidence for chemotaxis was demonstrated in A. caulinodans, it may be possible to do so in the future with aforementioned optimisation.

References & Attributions

Attributions: Connor Trotter and Sadiya Quazi

1. Liu W, et al. (2017) Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems. Frontiers in Microbiology 8:1327.

2. Pedrosa FO, et al. (2011) Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses. PLoS Genetics 7(5):e1002064. 261.

3. Lee K-A, Moon SH, Kim K-T, Mendonca AF, & Paik H-D (2010) Antimicrobial Effects of Various Flavonoids on Escherichia coli O157:H7 Cell Growth and Lipopolysaccharide Production. Food Science and Biotechnology 19(1):257.

4. Pham HT & Parkinson JS (2011) Phenol Sensing by Escherichia coli Chemoreceptors: a Nonclassical Mechanism. Journal of Bacteriology 193(23):6597-6604.

5. Dreyfus BG, JL; Gillis, M (1988) Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a Stem-Nodulating Nitrogen-Fixing Bacterium Isolated from Sesbania rostrata. International Journal of Systematic Bacteriology 38:89-98.

6. Tarrand JJ, Kried NR, Doebereiner J (1978) A Taxonomic Study of the Spirillum lipoferum Group, with Descriptions of a New Genus, Azospirillum gen. nov. and Two Species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Canadian Journal of Microbiology 24: 967-980.

7. Baldani JI, Baldani VLD, Seldin L, Doebereiner J (1986) Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a Root-Associated Nitrogen-Fixing Bacterium International Journal of Systematic and Evolutionary Microbiology 36: 86-93, doi: 10.1099/00207713-36-1-86.

8. Tadra-Sfeir MZ, et al. (2015) Genome Wide Transcriptional Profiling of Herbaspirillum seropedicae SmR1 Grown in the Presence of Naringenin. Frontiers in Microbiology 6:491.

9. Reyes-Darias JA, García V, Rico M, Corral-Lugo A, & Krell T (2016) Identification and Characterization of Bacterial Chemoreceptors Using Quantitative Capillary and Gradient Plate Chemotaxis Assays. Bio-protocol 6(8):e1789.

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-                 <p>In order to utilise microbe-microbe signalling in order to manipulate the microbiome, it was important to understand the response said chemical signal elicits. We examined how three species of free-living nitrogen-fixing bacteria respond to the presence of our selected flavonoid - naringenin. The three species chosen, Azorhizobium caulinodans (ORS571), Azospirillum brasilense (SP245), and Herbaspirillum seropedicae (Z67), were selected as they all have potential to form different types of interactions with plant roots. A. caulinodans has been shown to fix nitrogen both as a free living microbe and when in symbiosis with Sesbania rostrata, a semi-aquatic tree [1]. H. seropedicae  is a root endophyte, much like the  Pseudomonas sp. we investigated and frequently colonises popular crops such as wheat and maize [2].  </p>
+                 <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>The three free-living nitrogen-fixing bacteria used as part of our project were new to our laboratory; thus we needed to understand their growth characteristics before commencing chemotaxis studies. As such, the first area we studied was colony morphology. The results from this study served as a control to which experimental plates were compared to. Familiarisation with the bacteria also allowed identification of abnormal behaviour and contamination.</p>
+               <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>, and <i>Herbaspirillum seropedicae</i> colonies grown on solid media.</font>
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            <td>White</td>
            <td>Regular form, Typically raised, Entire margin</td>
-           <td>Colonies rarely grow to a measurable size when grown at 30˚c on YEB media after 24 hours</td>
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            <td>Cream/Light Green</td>
            <td>Circular or Irregular form (occasionally rhizoid), Raised elevation, Shiny</td>
-           <td>Colonies took on a different morphology depending on how the media was innoculated; stab-innoculation lead to rhizoid form while spreading leads to circular/irregular form</td>
+           <td>Colonies took on a different morphology depending on how the media was inoculated; stab-inoculation lead to rhizoid form while spreading leads to circular/irregular form</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> Laboratory robotics and smart lab equipment provides the platform for high reproducibility in experiments, however due to their current price bracket are unavailable to the majority of labs. This has driven recent developments in making affordable technologies for lab equipment, notably in the release of the Opentrons OT-2 liquid handling robot and the smart incubator by Incuvers Incorporated. With these developments and the ever-growing library of software, the likelihood that BDA will be integrated into most modern labs is high. </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 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 1d). If the culture is spread across the plate, then it typically takes a circular or irregular form (Figure 1c). 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>
+             <img src="https://static.igem.org/mediawiki/2018/b/be/T--Newcastle--AllPreservePlatesNew.png">
+<p> </p>
+             <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-inoculated.  </font>
              </div>
@@ Line 159: / Line 163: @@
-     <section id='team' class="s-services">
+     <section id='GrowthinLiquidMedia' class="s-services">
          <div class="row section-header has-bottom-sep" data-aos="fade-up">
                  <div class="col-full">
-                             <h3 class="subhead">Minimal Information Standards</h3>
+                             <h3 class="subhead">Growth Rates in Liquid Media</h3>
                  </div>
@@ Line 172: / Line 176: @@
          <div class="row about-desc" data-aos="fade-up">
              <div class="col-full">
-                 <p>Minimum information standards (MIEO) provide explicit information on what information needs to be reported out of the experimental metadata that could influence the reproducibility of the result (Decoene et al. 2018). The following factors, based around Hecht and colleagues (2018) work (full MIEO in appendix), focused on experimental factors deemed most necessary in the growth and productivity of engineered organisms. This provides specific details for use of microtiter 96 well plates and shake flasks during culturing. </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 (Figure 2).</p>
-                <b style="font-size:30px"> Media Components </b>
+<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>
-                </div>
+<img src="https://static.igem.org/mediawiki/2018/e/e9/T--Newcastle--GrowthCurveGraphNewChemotaxisNonTrans.png">
-                    <b> Effects on Growth </b>
+<p> </p>
-                        <p> Growth media is essential to any form of microbial culture, providing the nutrition required for optimal growth. There are a number of different options available, with Lysogeny Broth (LB) (Bertani 2004), Super Optimal Broth (SOB) (Hanahan 1983) and Terrific Broth (TBr) (Tartof 1987) being the most commonly used. However most are rich and undefined media, containing extracts such as yeast or beef that have an unquantifiable and highly variable composition. These extracts are also generally more expensive, complicate recovery and, due to their variable composition, result in significant batch-to-batch variation (Lee 1996; Moser et al. 2012). In the literature researched, TBr contributed to the highest amount of culture growth with Escherichia coli, with Losen et al. (2004) stating that TBr lead to an increase of 5x biomass when compared to LB. Islam (2007) produced similar results, with a significantly higher soluble protein yield in TBr than LB. This was put down to having glycerol as a defined carbon source. Furthermore, it is suggested that glucose is a poor choice due to E. coli excreting acetic acid as a by-product of glucose consumption, lowering pH and reducing growth (Islam et al. 2007; Losen et al. 2004; Marini et al. 2014). Glucose however is not the only issue. Singh et al. (2017) suggests that the carbon and nitrogen source are the most important components of the media as they can affect the type and amount of product produced. Other studies have concluded that E. coli develops a media history, adapting to different medias over time, showing variations in ribosome and RNA polymerase efficacy due to the medias amino acid makeup (Ehrenberg et al. 2013; Paliy and Gunasekera 2007). </p>
+             <font size="2">Figure 2. Growth curves showing changes in absorbance at 600 nm of <i>E. coli</i>, <i>A. caulinodans</i>, <i>H. seropedicae,</i> and <i> A. brasilense</i> in LB at 30 °C for 70 hours. n=4 replicates, error bars indicate standard error of the mean.</font>
-                        <p> Inorganic ions can also play an important role in the growth of cultures. Studier (2005) carried out an exhaustive study on inducer effects in media, investigating a number of variables as well as the presence of inorganic ions. The data collected showed that phosphate promoted kanamycin resistance, while sulphate supported optimum growth. However, on the contrary limiting magnesium concentrations allowed the cell culture to grow to a higher OD600. </p>
-                    <b> Effects on Protein Yield </b>
-                        <p> It is known that with an increased amount of cell growth, there is generally a higher yield of recombinant protein (Khan et al. 2009). If E. coli are made to produce protein at too high a rate however, inclusion bodies will form which is deemed inefficient due to the complex process of refolding them into functional proteins (Marini et al. 2014). Marini and colleagues (2014) showed that even with higher cell density, functional protein expression had no change. Therefore, there must be a point of optimal cell growth that provides the highest amount of functional protein whilst causing the least amount of inclusion body formation or incorrect protein folding. Antibiotic selection can also impact on the protein yield. Using kanamycin in higher concentrations has been shown to increase plasmid stability and allow maintenance of higher plasmid copy numbers due to selection pressures (Kelly et al. 2009). </p>
-                <b style="font-size:30px"> Media Properties </b>
-                        <p> Control of pH is essential for growth mediums. All forms of bacterium have optimum pH’s, even at the extremes (acidophiles and alkaliphiles). However, in current synthetic biology many of the used chassis are generally classified as neutrophiles, so maintaining a pH of between 6-8 is essential. Presser et al. (1997) carried out an in-depth study of E. coli growth rates modelling the growth as a function of pH and lactic acid concentration. From this, E. coli was determined to have a pH boundary of 4.0, beyond which resulted in no growth. Lactic acid was found to be inhibitory in high concentrations, something required to consider during scale-up. This links back to aforementioned studies that showed the importance of carbon source, with glucose instigating a drop of pH and inhibition of E. coli growth (Islam et al. 2007; Losen et al. 2004; Marini et al. 2014). However, for very niche experimentation, suboptimal pH may play an important role in experimental design. Maurer et al. (2005) discussed how pH regulates a number of genes, including flagellar motility, catabolism and oxidative stress in E. coli. High pH 8.7 was found to repress membrane proteins, chemotaxis and flagellar motility. Low, acidic conditions of pH 5.0 were found to increase metabolic rates. In conclusion, experimentation entailing genetic circuits and protein production can be said to be drastically affected by pH. pH therefore must be defined in the experimental method for reproducible results. </p>
-                <b style="font-size:30px"> Container Geometry and Shaking </b>
-                        <p> If synthetic biology is going to follow a BDA approach, robotics will need to be implemented into microbial growth workflows. For high-throughput, this requires the use of multi-well plates and much smaller volumes than standard batch microbial growth techniques. This opens up an entirely new area of irreproducibility and standardising these experiments is therefore crucial to further understanding. Additionally, in a study of 49 papers in the field of synthetic biology it was estimated that upwards of 80% of papers did not provide complete information to fully reproduce their data (Chavez et al. 2017). </p>
-                        <p> In microtiter plates, there are two main variables that need to be optimised; the oxygen transfer rate (OTR) and in turn, the overall mass transfer (KLa). Both of these variables have a significant inhibiting effect on microbial growth if not optimised. Fortunately in microtiter plates, both variables can be optimised in tandem by employing the same methods. OTR has been found to increase with increasing well size and a decrease in fill volume, as would be expected due to the reliance of surface aeration (Running and Bansal 2016; Hermann et al. 2003; Schiefelbein et al. 2013). Hermann and colleagues (2003) presented a clear correlation that a decreasing fill volume results in an increased OTRmax, that is only accentuated with increasing shake speeds. However, increasing the media viscosity can also decrease the OTR (Giese et al. 2014; Klöckner et al. 2013; Running and Bansal 2016), so shaking and baffling are essential to the optimisation of growth. If multiple wells have different viscosities a compromise must be made. </p>
-                        <p> Shaking in microtitre plates needs to surpass the critical shaking frequency, whereby the centrifugal force exceeds that of the interfacial surface tension (Hermann et al. 2003; Kensy et al. 2005). Funke and colleagues (2009) states that below 500 rpm, this is not reached and no significant increase in OTR is seen. Unfortunately the data is not shown, but due to their significant OTR increases from 500-1000 rpm, this suggests it is reliable. The shaking diameter for their results only covers 3 mm, but previous work has conflicting results, using a larger shaking diameter and lower rpm OTR. In two papers, a 300 rpm and shaking diameter of 50 mm was shown to increase OTR significantly, with a shaking diameter of 25 mm showing a 3x decrease when compared with 50 mm (Duetz et al. 2000). However there was some splashing in larger wells (Duetz and Witholt 2004). On the contrary, Hermann et al. (2003) found that a shaking at 300 rpm at 25 mm produced no significant difference in OTR than if not shaken but also confirmed that any higher than 400 rpm at 25 mm would cause liquid spillage. </p>
-                        <p> Baffling changes the flow characteristics of wells, increasing the turbulence and mixing. In microtitre plates, baffling is not standardised as in shake flasks, so the amount of laboratory’s with access to intentionally baffled microtiter plates is limited. Baffling in microtitre plates can also increase the chance of ‘out-of-phase phenomena’, where the flow of liquid creates an unmixed space at the bottom of the well (Büchs et al. 2001). Funke et al. (2009) designed 30 different well shapes, with a gradually increasing number of edges/baffles. From this, standard spherical wells were found to have the worst OTRmax and KLa, whilst the novel 6 edged petal shape allowed maximal OTR. Realistically, the 6 edged petal shape used is not commercially available so it would be unacceptable to suggest this use is common place. Despite this, other research groups have found that square wells have a baffle-like effect (Duetz 2007; Duetz and Witholt 2004; Hermann et al. 2003). </p>
-                        <p> Wells are usually covered to prevent evaporation throughout experimentation. The most common are oils, lids, stickers and seals. The type of cover used can significantly affect the growth rates and overall experimental data (Chavez et al. 2017). Oil has been shown to prevent evaporation entirely, however it significantly lowers the OTR and reduces protein expression, making it sub-optimal for most synthetic biology uses (Chavez et al. 2017). Chavez and colleagues (2017) found that lid and sticker covers allowed for the greatest OTR and protein expression and that lid covering also caused the highest evaporation rate, specifically in the four corner wells. Sealing the plate is another option, however this method has only been to shown to reduce the OTR with minimal evaporation prevention (Zimmermann et al. 2003; Sieben et al. 2016). </p>
-                    </div>
     </div>       <!-- end services-list -->
+</section>
-     <section id='team' class="s-services">
+     <section id='NaringeninMIC' class="s-services">
          <div class="row section-header has-bottom-sep" data-aos="fade-up">
              <div class="col-full">
-                 <h3 class="subhead"></h3>
+                 <h3 class="subhead">Effect of Naringenin on Growth Rate in Liquid Culture</h3>
-                       <h1 class="display-2">Aims</h1>
+                       <h1 class="display-2"></h1>
                  </div>
@@ Line 215: / Line 200: @@
          <div class="row about-desc" data-aos="fade-up">
              <div class="col-full">
-                 <p> The overarching aim for the 2018 Interlab focuses on the weakness in the measurement of fluorescence relative to optical density (OD), as with previous IGEM interlab protocols there is 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 also allows 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>
+                 <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 <a href="https://2018.igem.org/Team:Newcastle/Results/Operon" class="black">naringenin biosynthesis operon</a> 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>
-                <p> However, in this case the GeneMachine team further investigated the core reproducibility and standardisation aspect of the Interlab. What were its flaws and weaknesses? How could variation be minimised? How could it be standardised? Using a statistically driven Design of Experiments (DoE) methodology to aid in optimisation and a BDA workflow for enhanced reproducibility and standardisation, three main aims were investigated: </p>
-                <ul style="list-style-type:circle;display:grid;text-align:left;">
-                    <li> The use of an internal standard to allow comparative results through all test devices indicating sources of variation in protein production and expression. This should highlight the efficacy of promoter strength and resulting protein production. </li>
-                    <li> The development of a E. coli Dh5a growth model to investigate how media effects the expression of the Interlab test devices and to determine the optimal media for reproducible results. </li>
-                    <li> The automation and optimisation of competent cell preparation and transformation workflows. To create an automated and most importantly robust protocol to allow the reproducible generation of competent cells for consistent transformation of E.coli Dh5a. </li>
-                </ul>
+<img src="https://static.igem.org/mediawiki/2018/d/d7/T--Newcastle--KillCurveGraphNewChemotaxis2.png">
+            <font size="2">Figure 3. 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. n=3 replicates, error bars indicate standard error of the mean. </font>
+            <p> </p>
+            <p style="font-size:medium">All species successfully grew in the presence of 0-150 μM naringenin (Figure 3). However, it was noted that when the concentration of naringenin exceeded 100 μM, the amount of error also increases. This suggests that naringenin begins to have a greater impact on some, but not all, bacteria in the solution. As such, naringenin concentrations of <100 μM were used as part of subsequent chemotaxis assays to avoid negatively impacting bacterial growth.  </p>
              </div>
@@ Line 235: / Line 214: @@
 </section>
-    <section id='team' class="s-services">
+<section id='CapillaryMethod' class="s-services">
          <div class="row section-header has-bottom-sep" data-aos="fade-up">
                  <div class="col-full">
-                            <h3 class="subhead"></h3>
+                    <h3 class="subhead"></h3>
-                 <h1 class="display-2">InterLab Protocol</h1>
+                 <h1 class="display-2">Characterising Chemotactic Behaviour</h1>
+                </div>
+        </div> <!-- end section-header -->
+<div class="row section-header has-bottom-sep" data-aos="fade-up">
+                <div class="col-full">
+                            <h3 class="subhead">Quantification Utilising Capillaries</h3>
                  </div>
          </div> <!-- end section-header -->
-        <div class="row services-list block-1-2 block-tab-full">
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-                 <p>Following calibrations, two transformed colonies for each test device and both controls were used to inoculate LB medium containing 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, plasmid backbone and protocol workflow are shown in figure 2.</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>
-<img src="https://static.igem.org/mediawiki/2018/4/4e/T--Newcastle--IntProt.PNG">
+<font size="2">Table 2: Colony forming units of four bacterial species from capillaries containing 1 µl 100 µM naringenin or chemotaxis buffer solution (10 mM potassium phosphate, 0.1 mM EDTA, 10 mM glucose, pH 7.0) after 45 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 chemotaxis buffer was non-significant for all species (P > 0.05). </font>
+                <table id="protocols">
-                </div>
+      <thead>
+        <tr>
+          <th>Species (Strain)</th>
+          <th>Colony Count (Naringenin)</th>
+          <th>± Standard Error</th>
+          <th>Colony Count (Control)</th>
+          <th>± Standard Error</th>
+          <th>Significant Difference</th>
+        </tr>
+      </thead>
+      <tbody>
+        <tr>
+          <td><i>A. caulinodans</i> (ORS571)</td>
+          <td>0</td>
+          <td>0</td>
+          <td>0</td>
+          <td>0</td>
+          <td>No</td>
+        </tr>
+        <tr>
+          <td><i>A. brasilense</i> (SP245)</td>
+		  <td>0</td>
+          <td>0</td>
+          <td>0</td>
+          <td>0</td>
+          <td>No</td>
+        </tr>
+        <tr>
+          <td><i>H. seropedicae</i> (Z67)</td>
+          <td>458</td>
+          <td>86</td>
+          <td>376</td>
+          <td>236</td>
+          <td>No</td>
+          </tr>
+        <tr>
+          <td><i>E. coli</i> (DH5α)</td>
+          <td>0</td>
+          <td>0</td>
+          <td>0</td>
+          <td>0</td>
+          <td>No</td>
+          </tr>
+    </table>
+    <p> </p>
+    <p style="font-size:medium">After 24 hours incubation at either 30 °C (<i>A. caulinodans</i> and <i>H. seropedicae</i>) or 37 °C (<i>A. brasilense</i> and <i>E. coli</i>), the number of colonies which grew on the LB agar plate was counted (Table 2). The results showed that of the four test bacterial species, only one was able to move into the capillary. This species was <i>H. seropedicae</i> which was able to move successfully into capillaries containing either the control (buffer solution) or the chemoattractant. This was demonstrated by the growth of colonies on LB agar from the contents of each capillary (Figure 4). Both methods of agar inoculation (spreading and pipetteing) lead to colony growth.</p>
+   <p style="font-size:medium">After counting colonies from the contents of both the control and naringenin capillaries, no significant difference between mean colony count of the two conditions was observed. The results therefore show no evidence for positive chemotaxis using this method. It should be considered, however, that <i>H. seropedicae</i> was the only species that demonstrated growth on agar, and therefore the only one able to enter the capillaries. We concluded that this methodology is not yet sufficiently optimised for our application and may be having a confounding effect upon chemotactic response. </p>
+    <img src="https://static.igem.org/mediawiki/2018/1/1e/T--Newcastle--HerbaspirillumseropedicaeCapillaryPlates.png">
+<p> </p>
+     <font size="2">Figure 4. 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 45 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 chemotaxis buffer after 45 minutes open-end submersion in bacterial solution. Plates were inoculated via streaking technique and  incubated for 24 hours at 30 °C.
+    </div>
              </div>
@@ Line 260: / Line 298: @@
 </section>
-     <section id='team' class="s-services">
+     <section id='Microscopy' class="s-services">
          <div class="row section-header has-bottom-sep" data-aos="fade-up">
                  <div class="col-full">
-                             <h3 class="subhead"></h3>
+                             <h3 class="subhead">Microscopy Observations</h3>
-                 <h1 class="display-2">Abs<sub>600</sub> analysis of each test device</h1>
                  </div>
@@ Line 274: / Line 312: @@
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              <div class="col-full">
-                <p>All test devices produced growth to OD600 reading in excess of 0.3, except test device (TD) 4. Despite lower growth than other transformants, TD4 produced the highest mean fluorescence reading of 79.1 a.u., as was expected as the strongest promoter of the Anderson collection (parts.igem.org/Promoters/Catalog/Anderson). Figure 3A and 3B show the colony 1 and colony 2 Abs600 values for the controls and each test device at 0 hours and 6 hours respectively. </p>
+<p style="font-size:medium"> An alternative method of observing chemotactic responses is through the use of microscopy. Brightfield microscopy allows direct observations of bacterial responses. This will allow comparisons of motility and morphology from our experimental data to that of the published literature that was used to underpin our first iteration of the <a href="https://2018.igem.org/Team:Newcastle/Modelling/Community" class="black">community model</a>. Using microscopy enables the development of a cell density:optical density index (CD:OD index), a method of converting the two values. This index was also used in the community model to adapt the growth curve data collected during bacterial characterisation in standard laboratory conditions.</p>
-<img src="https://static.igem.org/mediawiki/2018/8/85/T--Newcastle--ABS600.PNG">
+<p style="font-size:medium">The CD:OD index was produced utilising data collected from a haemocytometer. A haemocytometer is a specialised microscopy slide of a known volume, it also contains a grid at the centre. By counting the number of cells in 16 squares at the top left  and performing a series of <a href="https://2018.igem.org/Team:Newcastle/Protocols" class="black" >mathematical calculations</a>, we were able to determine cell density. By utilising a spectrophotometer, we were also able to take a reading of the optical density (600 nm) and thus link the two together (Table 3).</p>
+<p style="font-size:medium"><i>A. brasilense</i> was unable to be counted accurately. This was as the bacteria was difficult to view under the microscope due to human limitations, in addition to highly variable optical density readings but consistently low cell densities despite more than sufficient incubation times. This happened throughout all replicates and as such results for this species have been omitted. Exploration of why this occurred shall be work for the future with the potential alternative approach of utilising flow cytometry. </p>
+<font size="2">Table 3: Cell density (cells.ml<sup>-1</sup>) of  <i>A.  caulinodans</i>, <i>H. seropedicae</i> and <i>E. coli</i> at different optical densities</font>
+                <table id="protocols">
+      <thead>
+        <tr>
+          <th>Species (Strain)</th>
+          <th>Optical Density</th>
+          <th>Cell Density</th>
+          </tr>
+      </thead>
+      <tbody>
+        <tr>
+          <td><i>A. caulinodans</i> (ORS571)</td>
+          <td>1.00</td>
+          <td>3.14x10<sup>5</sup> </td>
+        </tr>
+        <tr>
+          <td><i>H. seropedicae</i> (Z67)</td>
+          <td>0.05</td>
+          <td>1.05x10<sup>6</sup></td>
+          </tr>
+        <tr>
+          <td><i>E. coli</i> (DH5α)</td>
+          <td>1.00</td>
+          <td>7.4x10<sup>8</sup></td>
+          </tr>
+    </table>
+    <p> </p>
+    <p style="font-size:medium">Utilising a haemocytometer to count cells also allows observations of cell morphology and behaviour. This was used to an advantage as it allowed us to explore whether our bacteria’s morphology aligns with the literature that was followed to produce the community model. It also enabled observations of motility which was important after theories that the species were no longer motile which is why only <i>H. seropedicae</i> showed movement into the capillary during the attempt to quantify chemotaxis. </p>
+    <font size="2">Table 4: Microscopy observations of diameter (µm), cell length (µm), and motility in <i>A. brasilense</i>, <i>A.  caulinodans</i>, and <i>H. seropedicae</i> on a haemocytometer at 40x objective compared to information in utilised literature.</font>
+                <table id="protocols">
+      <thead>
+        <tr>
+          <th>Species (Strain)</th>
+          <th>Length in Literature</th>
+          <th>Mean Diameter</th>
+          <th>Cell Length in Literature</th>
+          <th>Mean Cell Length</th>
+          <th>Motile (Y/N)</th>
+        </tr>
+      </thead>
+      <tbody>
+        <tr>
+          <td><i>A. caulinodans</i> (ORS571)</td>
+          <td>1.5-2.5(5)</td>
+          <td>1.9</td>
+          <td>0.5-0.6 (5)</td>
+          <td>0.5</td>
+          <td>No</td>
+        </tr>
+        <tr>
+          <td><i>A. brasilense</i> (SP245)</td>
+		  <td>2.1-3.8 (6)</td>
+          <td>2.0</td>
+          <td>1.0 (6)</td>
+          <td>0.7</td>
+          <td>Yes</td>
+        </tr>
+        <tr>
+          <td><i>H. seropedicae</i> (Z67)</td>
+          <td>1.5-5 (7)</td>
+          <td>2.4</td>
+          <td>0.7 (7)</td>
+          <td>0.7</td>
+          <td>Yes (Highly)</td>
+          </tr>
+          </table>
+    <p> </p>
+    <p style="font-size:medium">While completing microscopy evaluations, we utilised the ibidi µ-Slide III 3-in-1 Chemotaxis Microscopy Slide provided by our sponsors at ibidi. 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">here.</a>
                  </div>
@@ Line 285: / Line 405: @@
 </section>
-     <section id='team' class="s-services">
+     <section id='AgarChemotaxis' class="s-services">
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                  <div class="col-full">
-                             <h3 class="subhead"></h3>
+                             <h3 class="subhead">Chemotaxis on Agar</h3>
-                <h1 class="display-2">Fluorescein/OD<sub>600</sub> and MEFL/particle analysis</h1>
                  </div>
@@ Line 299: / Line 418: @@
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-                <p>The relatively poor growth and high fluorescence levels effectively cancelled each other out when readings were converted to Fluorescence per OD600 and MEFL per OD600 measurements, resulting in TD4 transformants producing the highest expression levels (figure 3.1). The high fluorescence and MEFL per OD600 reading for TD4 despite lowest growth suggests expression of TD4 is not fully representative of the relative promoter strength; expression levels are interdependent with growth rate, with higher growth rates expected to produce higher expression levels (Scott et al. 2010). Expected fluorescence levels based on relative promoter strength reported for the Anderson collection of promoters did not match entirely the results produced here. In particular, TD5 utilising the promoter J23104 was expected to be the second strongest but yielded only the fourth highest fluorescence reading of 22.14 and 21.42 for colonies 1 and 2 respectively. Similarly, the highest fluorescence reading was recorded by TD1 (expected strength: third), though this test device produced the widest range in fluorescence reading between the two colonies (r = 36.2), despite both colonies having the closest OD600 reading of any of the test devices (r = 0.007). In addition to the iGEM repository documentation for relative strengths of the Anderson promoter collection, previous literature has also demonstrated that the J23101 and J23104 promoters should have almost equal strength (He et al. 2017).</p>
+<p style="font-size:medium">Our third approach to understanding bacterial chemotaxis involved both qualitative and semi-quantitative analysis. Growth of the nitrogen-fixing bacteria on solid media was observed to understand chemotactic behaviour in response to naringenin. </p>
-<p>While TD5 appeared to underperform compared to promoter activity previously reported in the literature, subsequent sequencing of test devices revealed that colonies labelled as TD5 had in fact been transformed with the positive control device. This may have simply been the result of human error when pipetting or labelling over the process of the study. The consistence of TD5 underperformance across multiple replications of the study suggests that this occurred early on. The variation in expression visible is particularly alarming for the J23101 promoter, which has been proposed and utilised in the literature as a reference promoter to characterise relative strengths of other promoters as relative promoter units (Kelly et al. 2009).</p>
-<img src="https://static.igem.org/mediawiki/2018/b/b1/T--Newcastle--Fluorescence.PNG">
-                </div>
-            </div>
+<p style="font-size:medium">Multiple different variants of agar assays were conducted to optimise the methodology.</p>
+<p style="font-size:medium"><b><u>Method 1:</u></b></p>
+<p> </p>
+<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 quantitative 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 style="font-size:medium"><b><u>Method 2:</u></b></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 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>
+				       <table id="protocols">
+      <thead>
+        <tr>
+          <th>Species (Strain)</th>
+          <th>Growth Distance Towards Naringenin</th>
+          <th>Growth Distance Towards Control</th>
+        </tr>
+      </thead>
+      <tbody>
+        <tr>
+          <td><i>A. caulinodans</i> (ORS571)</td>
+          <td>3.70</td>
+          <td>4.01</td>
+        </tr>
+        <tr>
+          <td><i>A. brasilense</i> (SP245)</td>
+		  <td>7.32</td>
+          <td>7.10</td>
+        </tr>
+        <tr>
+          <td><i>H. seropedicae</i> (Z67)</td>
+          <td>5.87</td>
+          <td>5.66</td>
+          </tr>
+          <tr>
+          <td><i>E. coli</i> (DH5α)</td>
+          <td>8.33</td>
+          <td>7.67</td>
+          </tr>
+          </table>
+          <p> </p>
+          <p style="font-size:medium">Once again, no evidence of chemotaxis towards naringenin was observed in any species. It was noted that the ‘halo’ around <i>H. seropedicae</i> colonies on plates containing naringenin were constricted and more closely situated to the colony margin. Through consulting the literature, it was revealed that high concentrations of naringenin can repress genes involved in chemotactic behaviour in this species (8). This may provide an explanation as to why chemotaxis was not observed in this species.</p>
+<p style="font-size:medium"><b><u>Method 3:</u></b></p>
+<p> </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">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 50 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 inoculation (4). Bacteria were inoculated 15 mm (<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>
+		       <table id="protocols">
+      <thead>
+        <tr>
+          <th>Species (Strain)</th>
+          <th>Naringenin Response Index</th>
+          <th>Control Response Index</th>
+          <th>Chemotactic Response</th>
+        </tr>
+      </thead>
+      <tbody>
+        <tr>
+          <td><i>A. caulinodans</i> (ORS571)</td>
+          <td>0.478</td>
+          <td>0.473</td>
+          <td>Negative</td>
+        </tr>
+        <tr>
+          <td><i>A. brasilense</i> (SP245)</td>
+		  <td>0.552</td>
+          <td>0.515</td>
+          <td>Positive</td>
+        </tr>
+        <tr>
+          <td><i>H. seropedicae</i> (Z67)</td>
+          <td>0.625</td>
+          <td>0.5</td>
+          <td>Positive</td>
+          </tr>
+          <tr>
+          <td><i>E. coli</i> (DH5α)</td>
+          <td>0.472</td>
+          <td>0.493</td>
+          <td>Negative</td>
+          </tr>
+          </table>
+          <p> </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 was 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>
           </div>       <!-- end services-list -->
 </section>
-     <section id='team' class="s-services">
+     <section id='Conclusions' class="s-services">
          <div class="row section-header has-bottom-sep" data-aos="fade-up">
                  <div class="col-full">
                              <h3 class="subhead"></h3>
-                 <h1 class="display-2">Sources of variation in the InterLab study design</h1>
+                 <h1 class="display-2">Conclusions</h1>
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@@ Line 326: / Line 535: @@
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-                 <p>While the InterLab study is an effective way of gathering large sets of data surrounding the parts and protocols, it does not consider all sources of variation within datasets or the variability between data sets. As there are many factors which cause variability in microbial protein expression and productivity, some of these factors may be more favoured than others, leading to an overrepresentation of these in the metadata. </p>
+                 <p style="font-size:medium">After successfully characterising how <i>A. brasilense</i>, <i>A.  caulinodans</i>, <i>H. seropedicae</i> and <i>E. coli</i> behave in a laboratory environment through means of understanding colonies and growth rates, we began to explore bacterial chemotaxis toward naringenin. While it may be possible to observe this behaviour in a quantitative fashion via microscopy or microfluidic methods. These methods, from the data gathered from these series of experiments, require a higher level of optimisation than semi-quantitative based methods. </p>
+				<p style="font-size:medium">Importantly, we were able to successfully demonstrate chemotaxis of <i>A. brasilense</i> and <i>H. seropedicae</i> toward 50 µM naringenin, these results came from semi-quantitative agar-based assays. While no evidence for chemotaxis was demonstrated in <i>A. caulinodans</i>, it may be possible to do so in the future with aforementioned optimisation.</p>
                  </div>
-            </div>
-        <div class="row section-header has-bottom-sep" data-aos="fade-up">
-                <div class="col-full">
-                            <h3 class="subhead">Competent cell protocols</h3>
-                <h1 class="display-2"></h1>
-                </div>
-        </div>
-        <div class="row services-list block-1-2 block-tab-full">
-        <div class="row about-desc" data-aos="fade-up">
-            <div class="col-full">
-                <p>Even prior to the main cell measurement protocol, irreproducibility had a significant effect in the preparation of competent cells and successful transformation. While the recommended CCMB80 and transformation protocols were followed exactly, successful expression of transformants were not guaranteed. It took 3 weeks of constant run-throughs within our lab to gain a transformation efficiency (TrE) of 5.05 x 106 before we could even start the Interlab. As such, the competent cell and transformation process was further investigated through a Bio-design Automation platform that used a design of experiments methodology to optimise transformation buffer (TB) composition. Utilising our recently acquired OT-2 liquid handling robot (Opentrons, USA), a robust automated competent cell preparation protocol was developed.</p>
-                </div>
-            </div>
-        <div class="row section-header has-bottom-sep" data-aos="fade-up">
-                <div class="col-full">
-                            <h3 class="subhead">Recovery period</h3>
-                <h1 class="display-2"></h1>
-                </div>
-        </div>
-        <div class="row services-list block-1-2 block-tab-full">
-        <div class="row about-desc" data-aos="fade-up">
-            <div class="col-full">
-                <p>One factor that has been overlooked in the literature is the recovery period. Anecdotal evidence during all transformation protocols has indicated that the antibiotic resistance gene has a significant impact on how long the recovery time needs to be. For chloramphenicol, the widespread suggestion of a 1-hour recovery incubation for optimal TrE is quite simply incorrect, with a recovery incubation time of upwards of 2 hours being required for optimal TrE in our study. This is irrespective of volume, be it in 2 mL microcentrifuge tubes or 96 well plates. However, if using a 96 well plate format, this recovery period requires full optimisation due to its suboptimal OTR and KLa characteristics. With the additional growth inhibition of the majority of TB compositions, this recovery step needs to be fully optimised for optimal TrE. Super optimal broth with catabolite repression (SOC) is regularly used instead of SOB to enhance cells recovery, however as it includes glucose, this was not considered due to the potential inhibitory effects of increasing pH due to glucose metabolism (Islam et al. 2007; Losen et al. 2004; Marini et al. 2014). </p>
-<p>Experimental observation found ampicillin (AMP) did not require longer incubation. Chloramphenicol’s requirement for increased recovery time can be explained through its mechanism of action, inhibiting protein synthesis via the inhibition of peptidyl transferase (Schifano et al. 2013; Wolfe and Hahn 1965). AMP however inhibits cell wall synthesis via inhibition of transpeptidase. Unlike AMP resistance, which involves the synthesis and excretion of either β-lactamase or penicillinase (Drawz and Bonomo 2010), chloramphenicol resistance is acquired by the synthesis of chloramphenicol acetyltransferase which is not readily excreted (Shaw 1983). This is beneficial for generating libraries as it decreases risk of satellite colonies, but the resistance mechanism may take longer to form and confer sufficient antibiotic resistance. As a result, for a further optimised protocol, using a plasmid that confers AMP resistance would be beneficial to minimise the protocol time requirement. This would also decrease the safety risk as while CAM is a known carcinogen, AMP is not. </p>
-                </div>
-            </div>
-        <div class="row section-header has-bottom-sep" data-aos="fade-up">
-                <div class="col-full">
-                            <h3 class="subhead">Use of mut3GFP as a reporter</h3>
-                <h1 class="display-2"></h1>
-                </div>
-        </div>
-        <div class="row services-list block-1-2 block-tab-full">
-        <div class="row about-desc" data-aos="fade-up">
-            <div class="col-full">
-                <p>Even prior to the main cell measurement protocol, irreproducibility had a significant effect in the preparation of competent cells and successful transformation. While the recommended CCMB80 and transformation protocols were followed exactly, successful expression of transformants were not guaranteed. It took 3 weeks of constant run-throughs within our lab to gain a transformation efficiency (TrE) of 5.05 x 106 before we could even start the Interlab. As such, the competent cell and transformation process was further investigated through a Bio-design Automation platform that used a design of experiments methodology to optimise transformation buffer (TB) composition. Utilising our recently acquired OT-2 liquid handling robot (Opentrons, USA), a robust automated competent cell preparation protocol was developed.</p>
-                </div>
-            </div>
-        <div class="row section-header has-bottom-sep" data-aos="fade-up">
-                <div class="col-full">
-                            <h3 class="subhead">Competent cell protocols</h3>
-                <h1 class="display-2"></h1>
-                </div>
-        </div>
-        <div class="row services-list block-1-2 block-tab-full">
-        <div class="row about-desc" data-aos="fade-up">
-            <div class="col-full">
-                <p>Even prior to the main cell measurement protocol, irreproducibility had a significant effect in the preparation of competent cells and successful transformation. While the recommended CCMB80 and transformation protocols were followed exactly, successful expression of transformants were not guaranteed. It took 3 weeks of constant run-throughs within our lab to gain a transformation efficiency (TrE) of 5.05 x 106 before we could even start the Interlab. As such, the competent cell and transformation process was further investigated through a Bio-design Automation platform that used a design of experiments methodology to optimise transformation buffer (TB) composition. Utilising our recently acquired OT-2 liquid handling robot (Opentrons, USA), a robust automated competent cell preparation protocol was developed.</p>
-                </div>
-            </div>
-        <div class="row section-header has-bottom-sep" data-aos="fade-up">
-                <div class="col-full">
-                            <h3 class="subhead">Competent cell protocols</h3>
-                <h1 class="display-2"></h1>
-                </div>
-        </div>
-        <div class="row services-list block-1-2 block-tab-full">
-        <div class="row about-desc" data-aos="fade-up">
-            <div class="col-full">
-                <p>Even prior to the main cell measurement protocol, irreproducibility had a significant effect in the preparation of competent cells and successful transformation. While the recommended CCMB80 and transformation protocols were followed exactly, successful expression of transformants were not guaranteed. It took 3 weeks of constant run-throughs within our lab to gain a transformation efficiency (TrE) of 5.05 x 106 before we could even start the Interlab. As such, the competent cell and transformation process was further investigated through a Bio-design Automation platform that used a design of experiments methodology to optimise transformation buffer (TB) composition. Utilising our recently acquired OT-2 liquid handling robot (Opentrons, USA), a robust automated competent cell preparation protocol was developed.</p>
-                </div>
-            </div>
-        </div> <!-- end section-header -->
-         </div>       <!-- end services-list -->
 </section>
+</section>
+</section>
 <section id='team' class="s-services">
@@ Line 450: / Line 555: @@
 <br>
 <br>
-<h3 class="subhead">InterLab</h3>
+<h3 class="subhead"></h3>
-                 <h1 class="display-2">REFERENCES</h1>
+                 <h1 class="display-2">References & Attributions</h1>
              </div>
@@ Line 469: / Line 574: @@
                  <div class="col-full">
+<p class="about-para"><font size="2"><strong>Attributions: Connor Trotter and Sadiya Quazi<br>
+</strong><font></p>
+<p class="about-para"><font size="2">1.	Liu W, et al. (2017) Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems. Frontiers in Microbiology 8:1327.<font></p>
+<p class="about-para"><font size="2">2.	Pedrosa FO, et al. (2011) Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses. PLoS Genetics 7(5):e1002064. 261.<font></p>
+<p class="about-para"><font size="2">3.	Lee K-A, Moon SH, Kim K-T, Mendonca AF, & Paik H-D (2010) Antimicrobial Effects of Various Flavonoids on Escherichia coli O157:H7 Cell Growth and Lipopolysaccharide Production. Food Science and Biotechnology 19(1):257.<font></p>
+<p class="about-para"><font size="2">4.	Pham HT & Parkinson JS (2011) Phenol Sensing by Escherichia coli Chemoreceptors: a Nonclassical Mechanism. Journal of Bacteriology 193(23):6597-6604.<font></p>
+<p class="about-para"><font size="2">5.	Dreyfus BG, JL; Gillis, M (1988) Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a Stem-Nodulating Nitrogen-Fixing Bacterium Isolated from Sesbania rostrata. International Journal of Systematic Bacteriology 38:89-98.<font></p>
+<p class="about-para"><font size="2">6.	Tarrand JJ, Kried NR, Doebereiner J (1978) A Taxonomic Study of the Spirillum lipoferum Group, with Descriptions of a New Genus, Azospirillum gen. nov. and Two Species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Canadian Journal of Microbiology 24: 967-980.<font></p>
+<p class="about-para"><font size="2">7.	Baldani JI, Baldani VLD, Seldin L, Doebereiner J (1986) Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a Root-Associated Nitrogen-Fixing Bacterium International Journal of Systematic and Evolutionary Microbiology 36: 86-93, doi: 10.1099/00207713-36-1-86.<font></p>
+<p class="about-para"><font size="2">8.	Tadra-Sfeir MZ, et al. (2015) Genome Wide Transcriptional Profiling of Herbaspirillum seropedicae SmR1 Grown in the Presence of Naringenin. Frontiers in Microbiology 6:491.<font></p>
+<p class="about-para"><font size="2">9. Reyes-Darias JA, García V, Rico M, Corral-Lugo A, & Krell T (2016) Identification and Characterization of Bacterial Chemoreceptors Using Quantitative Capillary and Gradient Plate Chemotaxis Assays. Bio-protocol 6(8):e1789.<font></p>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<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>
-<p class="about-para"><font size="2">15. Gross, H. and J. Loper (2009). Genomics of secondary metabolite production by Pseudomonas spp.<font></p>
-<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>
-<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>
-<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>
-<p class="about-para"><font size="2">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.<font></p>
-<p class="about-para"><font size="2">20. Maheshwari DK (2012) Bacteria in Agrobiology: Plant Probiotics (Springer Berlin Heidelberg).<font></p>
-<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>
-<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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