Team:Newcastle/Results/Chemotaxis
Alternative Roots
Exploring Bacterial Chemotaxis
Characterising Naringenin Chemotaxis
An Introduction
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].
Bacteria Characterisation
Colony Morphology
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.
Table 1: Qualitative analysis of Azorhizobium caulinodans, Azospirillum brasilense, 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 YEB media 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 innoculated; stab-innoculation lead to rhizoid form while spreading leads to circular/irregular form |
Studying colony morphology was approached from 2 aspects: the colony size after a minimum of 24 hours and aesthetic (shape and pigmentation). When concerning Azorhizobium caulinodans (Figure 1), it was noted that colonies did not grow to a measurable size within 24 hours at 30˚C on YEB media. Colonies contained white pigmentation and were generally raised in elevation with an entire margin. Larger colonies rarely grew larger than 2mm whilst smaller colonies, which were much more numerous, could not be accurately measured.
Azospirillum brasilense (Figure 2) colonies were distinguishable by their distinctive orange/pink pigmentation; although both immature and dead colonies lacked this pigmentation. Older colonies became ingrained into the agar, making them harder to remove without damaging the agar. The 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 Tryptone Soya Agar was 3mm, making Azospirillum brasilense the fastest growing of our nitrogen fixing bacteria.
Interestingly, young Azospirillum brasilense colonies were shiny, round and with entire margins. These young colonies may have some pigmentation near the centre where the cells are more mature. This is contrast to older colonies which maintain a somewhat different phenotype as they lose their shine as well as wrinkle. Wrinkling can often lead to the loss of the round shape.
Finally, H. seropedicae colonies took on different forms depending on how the plate was inoculated. If the plate was stab-inoculated, the colony took a rhizoid appearance (Figure 3a). On the other hand, if the culture was spread across the plate then it would typically take a circular or irregular form (Figure 3b). Colonies possessed a green-cream pigmentation and were raised from the surface. Most colonies were shiny and typically 1.5mm in diameter after 24 hours at 30˚C.
Growth Rates in Liquid Media
Sadiya's text
Effect of Naringenin on Growth Rate in Liquid Culture
During the period of initial research for the Alternative Roots project, it was noted that naringenin possesses antimicrobial properties, particularly towards E. coli [3]. As E. coli (DH5α) was used as both a control in chemotaxis assays and as the organism in which our naringenin biosynthesis operon would be assembled, it was deemed important to characterise the effect of increasing naringenin concentrations on growth rate in LB media. We also took this time to investigate the same effect on our nitrogen fixing bacteria.
This information was essential in guiding the chemotaxis assays as it enabled an understanding of appropriate naringenin concentrations which would not have detrimental impacts upon cell health. If cell health was to be impacted, then there is potential for positive chemotaxis to not be evident as cell death may lead to the appearance of chemorepulsion. This is particularly true when applying the response index as a semi-quantitative measure of chemotactic response [4].
Figure 4: Change in absorbance at 600nm of 4 bacterial species (Azospirillum brasilense, Azorhizobium caulinodans, Herbaspirillum seropedicae, and Escherichia coli) after 24 hours of growth when grown in liquid media containing different concentrations of naringeninCharacterising Chemotactic Behaviour
Quantification Utilising Capillaries
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.
Abs600 analysis of each test device
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.
Fluorescein/OD600 and MEFL/particle analysis
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).
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).
Sources of variation in the InterLab study design
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.
Competent cell protocols
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.
Recovery period
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).
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.
Use of mut3GFP as a reporter
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.
Competent cell protocols
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.
Competent cell protocols
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.
InterLab
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