Team:Sheffield/project/science/growthcurves

	GROWTH CURVES

INTRO	LB	M9	DEFINED

Introduction and Aims

Our proposed reporter systems required an E. coli strain that allows the manipulation of intracellular iron levels via siderophore uptake. We chose to use the JC28 strain that has an entC knockout mutation, meaning that it is unable to produce enterobactin. In order to keep intracellular iron-levels of JC28 low while the strain still grows, we needed to test a range of media and varying iron concentrations.

We carried out growth curve experiments to determine the growth model of both the W3110 (Wild Type) and JC28 (mutant) E. coli strains. As part of this, we cultured the wild type and mutant strains and measured the growth of the cultures both indirectly, by measuring optical density (fig. 1), and directly, by counting the number of colony forming units (CFU) plated on agar plates (fig. 2).

The bacteria were grown in three different growth media; Lysogeny broth (LB), M9 minimal media and EZ defined media. This was necessary because we wanted to characterise the growth of our mutant over a range of iron conditions. It was hypothesised that the mutant would grow nearly as well as the wild type when iron is plentiful and it would not have to rely on siderophores for iron uptake, but would be significantly limited by low-iron conditions.

The growth curves behaving as predicted would confirm that our mutant is impaired in iron acquisition, which is to be expected if the mutant is unable to produce siderophores.

LB liquid media

In order to confirm the effect of iron on growth, we first grew both strains in iron-rich liquid media (LB).

1 ml from an overnight culture of W3110 and JC28 was diluted into 50 ml of fresh LB media at an OD₆₀₀ of around 0.01 and incubated at 37 °C and 200 rpm. Growth kinetics were monitored taking the OD₆₀₀ in 30 min intervals from the time of inoculation using 1 ml samples (see Fig. 1).

CFU counts were performed from the same growth curve experiment. 1 ml samples were taken at 30 min intervals and 3x 10 µl of dilutions from 10-1 to 10-6 were spotted on a LB agar plate ad incubated at 37 °C overnight. The average CFU of these spots were taken for each dilution (Fig. 2).

As predicted, only slight differences in growth between both strains were observed in liquid LB media (Fig. 1 and 2). Interestingly, at the same time point (T4.5) the size of the mutant JC28 colonies were significantly smaller than the Wild Type W3110 colonies (Fig. 3).

Figure 1. Growth curves of E. coli W3110 (wild type) and JC28 (mutant) in liquid LB media from OD₆₀₀

Figure 2. CFU counts of W3110 (wild type) and JC28 (mutant) grown in liquid LB media.

Figure 3. Comparison of wild type and mutant colony sizes from T4.5 on LB agar plates after being incubated overnight at 37 °C.

M9 media

We measured the growth of W3110 (Wild Type ) and JC28 (mutant) in M9 minimal media with a range of added iron concentrations. It was hypothesised that JC28 would grow at a similar rate to W3110 in the high-iron media, but be unable to grow as rapidly in low-iron media.

1 ml from an overnight culture of W3110 and JC28 was diluted into 50 ml of fresh M9 minimal media at an OD₆₀₀ of around 0.01 and incubated at 37 °C and 200 rpm. Growth kinetics were monitored taking the OD₆₀₀ in 30 or 60 min intervals from the time of inoculation using 1 ml samples (Fig. 4)..

No significant growth of W3110 or JC28 was observed after 5 hours at any of the tested iron concentrations. This experiment showed that M9 media was missing some nutrients that our strains require, leading us to investigate a richer defined medium where we could still control the level of iron.

Figure 4. Growth curves of E. coli W3110 wild type and JC28 (mutant) in liquid M9 minimal media from OD₆₀₀

Defined media

We investigated the growth of W3110 (Wild Type ) and JC28 (mutant) in EZ defined media with two added iron concentrations, 0 µM and 20 µM. It was predicted that JC28 would grow at a similar rate to W3110 in the high-iron media, but be unable to grow as rapidly in low-iron media.

1 ml from an overnight culture of W3110 and JC28 was diluted into 50 ml of fresh defined media, either with or without iron (III) chloride added to 20 µM, at an OD₆₀₀ of around 0.01 and incubated at 37 °C and 200 rpm. Growth kinetics were monitored taking the OD₆₀₀ in 30 or 60 min intervals from the time of inoculation using 1 ml samples (Fig. 5).

The growth curves in defined media (Fig. 5) show that W3110 (wild type ) is not significantly affected by the varying iron concentrations we tested. However, JC28 (mutant) demonstrated significantly reduced growth when grown under iron-limited conditions. This shows that JC28 is deficient in iron uptake.

Figure 5. Growth curves of E. coli W3110 (wild type) and JC28 (mutant) in liquid defined media from OD₆₀₀, with either added iron (III) chloride to 20 µM or without.