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
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.
Media ComponentsGrowth 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).
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.
Effects on Protein YieldIt 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).
Media PropertiesControl 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.
Container Geometry and ShakingIf 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).
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.
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.
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).
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).
Aims
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.
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:
- 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.
- 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.
- 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.
InterLab Protocol
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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