Difference between revisions of "Part:BBa K346001:Experience"

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Applications of BBa_K346001

Operation Characterization

We speculate that the threshold of MerR response can be also manipulated by controlling the concentration of MerR in cytosol. As with the bacteria in natural environment, the concentration of MerR is stabilized at a certain level. In order to verify the speculation, promoters from parts.igem constitutive promoter library with different strength were prefixed before BBa_B0034+MerR coding sequence to exogenously maintain MerR expression at different intensity (Fig.6). The sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. Furthermore, we used pSB1A2 and pSB3K3 as backbones because of their different copy numbers which could also result in different MerR expression intensity (Fig.7), which was confirmed by an additional experiment.

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Fig.6. The construction of the system used for MerR operation characterization. Promoters from the parts.igem with different intensities were selected, leading to varying MerR concentrations. The transcription of PmerT is controlled by the percentage of Hg-bound MerR dimer, thus the expression of GFP can reflex the concentration of Hg (II).

Fig.7. The strategy of backbones swiching. We used pSB3K3, a low-copy number backbone, and pSB1A2, high-copy, to differ the expression of merR.

We carefully tested every combination (the strength of constitutive promoter controlling MerR expression and the backbone copy number) under induction with a Hg (II) concentration gradient ranging from 10^-9M to 10^-5M. The concentration gradient of Hg (II) is: 0, 1E-9, 3E-9, 5E-9, 8E-9, 1E-8, 3E-8, 5E-8, 8E-8, 1E-7, 3E-7, 5E-7, 8E-7, 1E-6, 3E-6, 5E-6, 8E-6, 1E-5, 3E-5, and 5E-5. Cell culture was cultivated for 8 hours and then dilute with fresh LB in a ratio of 1:100 and continue to incubate until the OD600 reached 0.4~0.6.

The result is shown in Fig.8.

Fig.8. The expression intensity of MerR significantly determines the threshold of sensitivity to mercury (II). Five representative lines are selected and it can be seen that the thresholds have varied apparently. The letter in the bracket after the promoter name denotes the backbone (pSB3K3 or pSB1A2) where Pc-RBS-merR was cloned. The deeper the colour, the stronger the expression level of MerR is, leading to a higher threshold.

The sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. Data demonstrates that cells with different MerR intensity exhibited correspondingly different sensitivity to mercury, indicating that the stronger the expression level of MerR is, a higher threshold is represented.

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