Team:Exeter/Improve

D. aromatica cld : BBa_K2695010

This part codes for the enzyme Cld from Dechloromonas aromatica to catalyse the second step of our enzyme pathway to reduce perchlorate to oxygen. Chlorite dismutase catalyses the reduction of a chlorite ion into a chloride ion and oxygen. In 2016 Leiden's ‘E. coliniser’ project added D. Aromatica cld part (BBa_k1938004) to the registry of standard parts. Through email correspondence we discovered that they had difficulty building their final construct and were therefore unable to characterise it. They were planning to insert the cld into one plasmid with the pcrABCD operon and an additional three genetic elements.

Due to the pcrABCD being such a large operon we decided to insert the operon into one plasmid and construct the D.aromatica cld with a T7 inducible promoter in a seperate but compatible plasmid. Splitting up the part made it easier to build and we were successful in protein expression and in assaying activity.

The successful cloning of Cld was shown by gel electrophoresis (Figure 1), expression was confirmed with a Western blot (Figure 2), and by measuring oxygen production using a Clark electrode we demonstrated enzyme activity (Figure 3). More detail on how each of these assays were performed can be found here.

Figure 1: Gel electrophoresis after a restriction enzyme digest of the chlorite dismutase constructs with EcoRI and PstI .

Figure 2: Western blot for chlorite dismutase constructs
Expected molecular weights (kDa): A = 32.97 | B = 32.99 | C = 32.96 | D = 31.20

Figure 3: Clark electrode data showing rate of oxygen production from four chlorite dismutases and a negative control.

The rate of oxygen production from the Clark electrode (Figure 3) is evidence that the enzyme worked as expected. D. aromatica would be the source of our first choice of chlorite dismutase based on this evidence.

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