Part:BBa_K1895999:Experience

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The overnight cultures of E. coli cells containing our BBa_K1895999 were diluted down to a starting optical density of around 0.05 at 600nm using LB broth with chloramphenicol. For characterisation of this device, we used a 96 well plate and used half of the plate to contain 3mM of ZnSO4 with 0.5% of arabinose and LB broth with chloramphenicol. The other half of the plate contained the 0.5% of arabinose with LB and chloramphenicol.The cells were then left to grow for 16 hours and the OD600 was measured every 5 minutes with orbital shaking ocuring inbetween.

<figure> <img alt="Zinc Growth" src=""/>

<figcaption>Figure 1. The minimum inhibitory concentration for E. coli growing in ZnSO₄. The cells were left for 16 hours to grow in different zinc sulphate concentrations with LB broth and chlormaphenicol. OD600 was measured after this 16 hour growth period.</figcaption>

<figcaption>From this experiment, we found that the minimum inhibitory concentration was 3mM of ZnSO₄ for E. coli. This concentration was then used in our second experiment to test whether our “Variable Resistor” (BBa_K1895999) construct was able to sequester zinc in the presence of arabinose.</figcaption>

</figure>

From our experiments, we can conclude that our ‘Variable Resistor’ (VR) construct does not grow in ZnSO4 when arabinose is present. The cells only grew when zinc was not present. Therefore, showing that our construct doesn’t sequester zinc when the SmtA protein is produced.