Introduction and Aims

We set out to form an understanding of the growth model of both the wildtype – W3110 – and mutant – JC28 – E. coli strains. The JC28 mutant has an entC knockout mutation meaning that it is unable to produce siderophores. JC28 genotype: W3110 ∆fecABCDE ∆zupT ∆mntH ∆entC ∆feoABC In order to do this we cultured the wildtype and mutant strains and measured the growth of the cultures both indirectly, by measuring optical density, and directly, by counting the number of colony forming units (CFU).

It was important to understand the growth rate of the two strains for a number of reasons:

1. When carrying out later experiments on either strain we will need to know how long to incubate the cultures for so that the growth of the colony is in the logarithmic phase. Beyond the log phase (stationary phase) the bacteria will start to lyse as the nutrients runs out and the bacteria start to starve. Before the log phase (lag phase) too few bacteria will be present and they will not be dividing at their optimum rate. Tests carried out on the bacteria in lag or stationary phases would not give representative data.
2. The entC mutation could have had effects on the growth rate of the mutant JC28 strain. In order to assess whether the mutant had a growth defect, we needed to compare the wildtype and mutant growth curves. Knowing if the mutant had a growth defect would allow us to take this into consideration in future experiments.

The Different Media

The bacteria were grown in three different growth media; Lysogeny broth (LB), M9 minimal media and EZ defined media. LB is a nutritionally rich medium that allows for rapid growth of E. coli. M9 minimal media contains the minimum set of nutrients for the growth of many wildtype E. coli strains. The EZ defined media contains only a specific set of salts, amino acids, and a carbon source. The defined media allows more precise control over the amount of iron available to the cells.

It was predicted that when both strains were grown in the LB liquid media there would be very little difference in the growth curves. This result is expected due to the LB media containing a good source of iron, ensuring that iron availability is not a limiting factor to growth.

However, it was hypothesised that in the defined media, the mutant bacteria would struggle to grow in low iron conditions and this would be reflected in a much lower growth curve. As an important element for many metabolic processes and ultimately for growth, the ability of the bacteria to scavenge and acquire iron becomes very important. The JC28 mutant does not produce siderophores – a key mechanism for iron scavenging and uptake – so their growth would be significantly inhibited when grown under iron-limited conditions.

LB liquid media

As predicted, there is only a slight difference in the growth curves of the two strains cultured in LB liquid media.

Figure 1. The optical densities of W3110 and JC28 E. coli strains at 30 minute intervals from the time of inoculation. The cultures were incubated in a 37OC environment and were suspended in a LB liquid media. 1ml samples were taken for each strain and the optical density at 600nm was recorded. W3110 is the wildtype E. coli whilst JC28 is the mutant E. coli lacking the functional entC gene. The JC28 mutant showed some slight growth defects.

Figure 2. Bacterial count (in colony forming units) from the point of inoculation. The cultures were incubated in a 37OC environment in LB liquid media. 1ml samples were taken at 30 minute intervals and 5 serial dilutions made. Each dilution was spotted 3 times onto a LB agar plate, the average CFU of these spots were taken for each dilution.

At the same time point (T4.5) the size of the mutant JC28 colonies were significantly smaller than the wildtype W3110 colonies as shown in Image 1 and 2.

M9 media

We measured the growth of W3110 and JC28 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.

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

Defined media

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