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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 and on the protocols page.
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 cultures over-expressing HpaC were tested, the absorbance levels were found to start much lower than the control. 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. Further tests will use differing amounts of cell lysate in order to try to capture data that shows the drop in absorbance for HpaC cultures.
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