Team:Tuebingen/Characterized Part

GLP.exe Characterized Part

Characterized Part


We characterized BBa_K398108, a salt tolerance cluster, for its resistance to potassium chloride. This was interesting to us because it is known that high obesity, a risk factor of Diabetes, correlates strongly with high salt intake [1]. Adding such a salt tolerance cluster to a probiotic therapeutic could provide it with a selective advantage, supporting the survival of the competition with other bacteria within the intestines. While the salt tolerance to sodium chloride was already characterized, the tolerance to potassium chloride was not. However, since this is one of the salts mainly found within the environment, we decided to characterize the part further.

BBa_K398108 salt tolerance cluster does not confer to an increased tolerance to potassium chloride.

We inoculated overnight cultures of DH5alpha wild type and salt tolerant DH5alpha in LB medium containing 0M to 0.8M KCl. Data was gained in triplicates and growth was measured as OD600 with a Nanospectrometer (Implen) every 45 minutes (in accordance with the previous characterization for NaCl) (Fig. 1). For the analysis, OD600 values were transformed to logarithmic values and subsequently nonlinearly fitted to a growth curve using GraphPad Prism8.0 (Fig.2).

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Figure 1: Mean of the triplicates and standard deviation of the measured growth curves.
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Table 1: Result of transformation to logarithmic scale and nonlinear fit to exponential population growth via Graph Pad Prism8.0. R squared is in the range of 0.89 to 0.96 for concentrations from 0M to 0.4M. R squared for 08M in the range of 0.1.
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Figure 2: Growth rate [h-1] and [%] growth of salt tolerant E. coli DH5alpha and wt DH5alpha. Growth rate was calculated by using the proposed doubling time of the fit. [%] growth was calculated by the normalisation of the growth rate of the salt tolerant DH5alpha against the wild type DH5alpha. Results obtained from triplicates for each concentration of potassium chloride.

The wild type DH5alpha showed approx. 20% higher growth rates compared to the salt tolerant strain in all compared concentrations, except for 0.8M KCl (Fig. 1), where the reduction of DH5alpha wild type was stronger.

Additionally, we expressed growth rates in [%] growth by normalizing the growth rates of DH5alpha with salt tolerance against the wild type growth rates. When looking at [%] growth, one may observe a slight increase in [%] growth of the salt tolerant DH5alpha upon addition of KCl. This may indicate that the salt tolerant strain is less inhibited in their growth, however these findings were not significant. To summarize, this suggests that the salt tolerance cluster does not confer to significant KCl tolerance in DH5alpha cells.