Part:BBa_K914000:Experience

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Applications of BBa_K914000

the supD gene rescues the kanamycin resistance at any concentration of the antibiotic. It shows also that there is no disadvantage to use the supD amber suppressor compared to the wild type kanamycin gene resistance, since there is no difference of growth rate (Figure 4B and 4C). However the leakiness of the Kan* gene is higher than expected, and thus one mutation is not sufficient for the containment. Indeed, at usual concentration, Kan* gene manages to express an antibiotic resistance, even though lower than Kan^R or supD (Figure 4A). Here we define the growth rate as the OD₆₀₀ observed at a given time (here t=8h20'), in order to overcome the fact that Kan*+RFP does not have a clear exponential phase. The fact that the strain (3) grows faster and can have higher OD₆₀₀ may be explain by the fact that supD is deleterious for the strain, by removing some stop codon of other genes for instance.

Figure 4A and 4B: The bar graph represent the OD₆₀₀ at time = 8h20' (black line Figure 4C) for the different strains at a given kanamycin concentration (black line on Figure 4B). On B, we observe the variations of the OD₆₀₀ at different kanamycin concentrations, at the same time.

Figure 4C: The variations of the OD₆₀₀ is observed in function of the time, for different concentrations of kanamycin (400µg/mL, 100µg/mL, 25µg/mL).

This part has been improved by part BBa_K2725016 - supD expressed under J23108 anderson promotor (strength 0.51). This expression level of supD allows the expression of a gene with 5 serine codons substituted for amber STOP codons.

File:T--Edinburgh UG--400.png

Figure 1: Growth curves of Top10 E.coli transformed with supD and recoded P1003 construct in 400 µg/mL Kanamycin.

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