Part:BBa_M36533:Experience

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Applications of BBa_M36533

B. Experiments that determine whether or not our designed actuator exhibits desired performance characteristics in S. cerevisiae

Cell Density Functional Assay

We conducted a functional assay with five arsenate concentrations from 12 ppb to 120,000 ppb and at five different time points for each of these two plasmid constructs. In this assay, our negative control was two replicates of transformed cells without arsenate, which demonstrates how the cells naturally grew in the 96-well plate. Two replicates of transformed cells with arsenate and without plasmid induction or repression provides a baseline of the cells’ intrinsic arsenic resistance as well as the toxicity of the plasmid itself. The two experimental conditions were transformed cells with arsenate which were repressed with 3% glucose and the plasmids induced by 3% galactose.

PHO84 Plasmid

The intended function of the PHO84 gene construct is to increase arsenate uptake into the cell. Since this gene has not been found to confer arsenate or arsenite resistance, our hypothesis was that the OD600 of the cells with the galactose-activated promoter would be lower than those with the glucose-repressed promoter. The cells with the glucose-repressed promoter should be comparable with the baseline of transformed cells with only arsenate.

However, at all concentrations and over time for all experimental groups, the observed trend was not consistent with the expected trend. After one hour, the cell density was highest for the cells exposed to 120,000 ppb; the cells with the galactose-induced promoter were higher than the negative control. After twenty hours of growth, the galactose-induced samples were higher in cell density than the glucose-induced samples in 40% of the concentrations (12 ppb and 120,000ppb).

http://i68.tinypic.com/r01t6o.png

Ultimately, the test was inconclusive. We have two possible explanations for this failure. First, we hypothesize that there could be an error in preparing and/or aliquoting arsenate stock concentrations for the samples. For example, OD600 at 1200 ppb is consistently higher than 120 ppb and thus, may have a lower arsenate concentration than stated. Second, we observed 10-fold differences in the OD600 for some replicates, suggesting spillage could have happened when transferring cells from the shaker to the plate reader and vice-versa. Third, it is possible that a 3% glucose solution was insufficient to completely repress the promoter; as a result, PHO84, coding for increased arsenic absorption, was still overexpressed. For example, in the figure below, the galactose-induced and glucose-repressed samples followed similar values and trends over time.

http://i65.tinypic.com/2128xv9.png

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