Part:BBa_K5109023:Experience

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Applications of BBa_K5109023

After successfully cloning Dehalogenase II inside pJUMP29-1A ∆NdeI (BBa_K5109060), before testing our part on PFAS molecules, we performed a control enzymatic test. Dehalogenase II is known to successfully degrade fluoroacetic and chloroacetic acid, therefore we decided to set some tests that would use chloroacetic acid in order to test the correct functioning of the protein: the chloroacetate substrate can be converted into glycolate after the removal of the chlorine done by the dehalogenase. We tested both wild type TOP10 F' as a control and TOP10 F' cells containing our expression vector. A part of the cells containing the vector were grown in addiction with IPTG 0.5 uM to induce protein expression, meanwhile another part was grown without IPTG as a control. All the cells were then incubated with LB medium + chloroacetic acid 2.5mM, and monitored for 24h. After the 24h we obtained the following results:

- F’ are wild type cells

- D9 are DeHa2 induced cells

- D9 NI are DeHa2 not induced cells

After 24h, the concentration of chloroacetic acid the medium decreased for 98% in cells that were induced with IPTG, while chloroacetic acid was degraded of 90% in cells without IPTG. As expected, wild type cells did not show any particular change in chloroacetate concentration over time Colonies expressing DeHa2 showed degradation both under IPTG induction and without IPTG: this may suggest a possible leaky activity of IPTG promoter.

The results lead us to consider part K5109023 a possible display system for the expression of an outer membrane protein. Further investigation is needed in order to confirm the correct functioning of the protein, and to subsequently test it on PFAS molecules.

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