Part:BBa_K902058:Experience

Experience

HpaC activity was tested by comparing cell lysate ability to consume NADH between cultures over-expressing HpaC to a control culture that is not. This is done by monitoring the decrease in absorbance at 340 nm (Kamali et al., 2010). In order to do this, cultures of pLacI-hpaC and pLacI-dszB were grown up overnight in LB with appropriate antibiotics. Following this, protein expression was induced with IPTG, after which the assay was carried out as described in the following figure.

Figure 1: HpaC Assay with A) 2 mL cell lysate and B) 100 µL cell lysate. Cultures of pLacI-hpaC and pLacI-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 µM IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 µM NADH and 20 µM FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. pLacI-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the pLacI-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.

When the assay was run, it was found that NADH does not convert readily to NAD+ on its own. When cell lysate containing the naturally expressed amounts of oxidoreductase was added, a decrease in absorbance could quickly be observed as the NADH was converted to NAD+. When cell lysates from 2 mL cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control (See figure A). We believe that this is because with the amount of cell lysate tested, when the HpaC protein is overexpressed the NADH is consumed almost immediately and therefore the data reflecting the drop in absorbance is missed. When 100 µL of cell lysate is used instead, the sharp decrease in absorbance due to the consumption of NADH can be observed for cultures overexpressing HpaC (See figure B). When contrasted to the rate of the control expressing native amounts of oxido-reductase, it is clear that HpaC is functional.

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