Part:BBa_M36534:Experience

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Applications of BBa_M36534

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.

ACR12 plasmid

The intended function of the ACR12 gene construct is to reduce arsenate to arsenite and to confer arsenite resistance. Because this gene construct increases cell resistance, we hypothesize that I) cell density should decrease for all conditions as arsenate concentration increases II) at a given arsenate concentration, the OD600 of cells with the galactose-activated plasmid would be higher than those with the glucose-repressed plasmid and the baseline.

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As shown the graph above, at one hour, the OD600 was much higher at the higher concentrations of arsenate than the lower concentrations, challenging hypothesis I. We hypothesize that this unexpected reading could have been due to the fact that during sample preparation, we loaded the lowest concentrations of arsenate first and had a 5-7 minute difference between the time arsenate was loaded into the 12 ppb well and the 120,000 ppb well. According to Shen, arsenate uptake into cells occurs quite rapidly, often within 10 minutes; however, the ACR genes often take two or more hours to fully take effect.4 As a result, more cells at the lower concentrations could have died before we put the plate in the shaker to grow.

The data were consistent with the expected trend for hypothesis II for both control and experimental groups and at all arsenate concentrations. After 2 hours of growth, the OD600 for different arsenate concentrations started to level off, with the galactose-induced samples having a highly statistically significant (p = 1.4 *10-5) difference from the baseline. We hypothesize that this may be the timepoint where the gene is expressed and starts reducing arsenate and conferring arsenite resistance.

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By the next morning (15-20 hours), both the general trends of hypothesis I and II were supported by the data; however, the difference between the galactose-induced promoter and the baseline was no longer statistically significant (p=0.413). We hypothesize that the difference is no longer statistically significant due to the saturation of the ACR12 gene complex in converting arsenate to arsenite before 15 hours.

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