Team:Newcastle/Proof/VariableResistor

Results for Arabinose controlled variable resistor

Growth in Varying ZnSO₄ Concentrations (Minimum Inhibitory Concentration)

In this experiment, we used 6mM of zinc sulphate (ZnSO₄) and carried out a two fold serial dilution for each row of a 96 well plate to try and identify the minimum inhibitory concentration for the E. coli to grow in. Constructs were again placed into the pSB1C3 backbone, cloned into E. coli and fresh cultures were grown at 37°C overnight. The cells were diluted down to a starting optical density of around 0.05 at 600nm using LB broth with chloramphenicol. Then, 100μl of cells were placed in the wells with the corresponding ZnSO₄ concentration which was diluted using LB with chloramphenicol. This experiment was left for 16 hours to give the E. coli time to grow and replicate, see figure 1.

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.

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.

Growth with Zn and Arabinose

The minimum inhibitory concentration was used for this experiment to determine whether the construct would sequester the zinc if arabinose was present. For this, 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 grown up in 10ml of liquid culture of LB broth with chloramphenicol overnight at 37°C. The cells were then diluted down to an OD of 0.05 at 600nm and 100μl of the cells were then added to each well. The cells were then left to grow for 16 hours and the OD600 was measured every 5 minutes. The results can be seen below in figure 2.

Figure 2. Growth curves of E. coli. Those growing with zinc were grown in 3mM of ZnSO4 with 0.5% arabinose and LB broth with chloramphenicol. Those growing without zinc were grown in 0.5% arabinose and LB broth with chloramphenicol. The OD600 was measured every five minutes, and the cultures were shaken in between the readings. The cells were grown for 16 hours. VR = Variable resistor BBa_K1895999, K1895100 = Our positive control BBa_K1895100, WT= Wild-type

Conclusion

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.