Part:BBa_K2695002

N. defluvii chlorite dismutase

Usage and Biology

This BioBrick is included in our chlorite dismutase parts collection along with BBa_K2695000, BBa_K2695001 and BBa_K2695010.

Chlorite dismutase is an enzyme used mainly by perchlorate reducing bacteria to break down the anion chlorite into chloride and oxygen - a reaction which takes place in the periplasm of the bacterium. Chlorite is damaging to cells as it is a strong oxidant, so even though the reaction it catalyses does not release energy it is essential for survival of these organisms. No other enzymes (except for photosystem 2) are known to have the primary function of producing oxygen through a covalent oxygen-oxygen double bond. (Kostan et. al., 2010).

This part was taken from the nitrite oxidising bacterium Nitrospira defluvii. This species is gram-negative and belongs to the phylum Nitrospirae, meaning it is only distantly related to other species of bacteria who derive energy from oxidising nitrite to nitrate (Maixner et. al., 2008). Interestingly, it contains a gene similar to the ones that code for the enzyme chlorite dismutase in perchlorate / chlorate reducing bacteria (PCRB) (Maixner et. al.,. 2008). However, its chlorite dismutase is found to be active in the presence of oxygen (cld only works in anaerobic conditions in PCRB (review article), and nitrite oxidising bacteria cannot use perchlorate as an electron acceptor when reducing nitrite (Kostan et. al., 2010). However, the enzyme has been shown to be expressed and active in E. coli as both chloride and oxygen were detected on the addition of chlorite to transformed cells (Maixner et. al., 2008).

Exeter iGEM 2018 chose to use the coding sequence for the chlorite dismutase enzyme from N. defluvii because of the kinetics (reference) compared to that of D. aromatica, A. suillum and A. oryzae. It was not expected to be as effective (as the other 3 enzymes) at reducing chlorite in E. coli, due to the fact that it isn’t from a species capable of deriving energy from perchlorate reduction.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 8
  Illegal BsaI.rc site found at 861
  

Cultivation and Expression

Cultivation:

• Cells were incubated overnight in 5ml LB media (35μg/ml chloramphenicol) at 37^oC shaking at 220 rpm.

Expression:

• Cells were grown in 50ml LB media (35μg/ul chloramphenicol) to an OD₆₀₀ of 0.6, verifying via a spectrophotometer.
• 0.2mM IPTG was added
• 1mM Hemin was added
• Cells were grown for a further 4 hours before harvesting

Western Blot

To demonstrate visually that the proteins were expressed a Western Blot was performed. The Blot membranes were probed with an anti-His-tag primary antibody raised in mouse, and an anti-mouse secondary antibody, raised in goat, conjugated to alkaline phosphatase.

                                     Figure 5: Western blot for Cld constructs 
                  Expected molecular weights (kDa): A = 32.97 | B = 32.99 | C = 32.96 | D = 31.20

The western blot does not show expression of chlorite dismutase for N. defluvii.

Cell Lysate Oxygen Production

Oxygen production was chosen as the parameter for enzyme activity, and this was measured using a Clark oxygen electrode. The experiment was performed using lysed, rather than whole, cells to get a 'yes or no' answer to the question 'will oxygen be produced on the addition of sodium chlorite?'

Harvested cells were resuspended in a phosphate buffer, pH 7 and lysed via sonication. Evolution of oxygen from the cell lysate, after addition of sodium chlorite (250 μM), was measured using a Hansatech Clark Oxygen Electrode.

NOTE: Sodium chlorite is extremely toxic (it is a suspected carcinogen) - because of this the oxygen measurement was carried out by a trained supervisor, NOT a member of the iGEM team.

                                         WT = Wild Type E. coli (strain: BL21 (DE3))

No oxygen was detected for BL21 (DE3) cells transformed with N. defluvii Cld on the addition of chlorite. Therefore it can be concluded that N. defluvii cld is not active in E. coli, or that it did not metabolise chlorite fast enough to produce levels of oxygen that could be detected by the electrode. Out of the parts collection of chlorite dismutase enzymes, N. defluvii cld was the least effective at producing oxygen.

The results of this experiment lay the groundwork for future repeats using whole cells to test whether the oxygen will be released from the periplasm of the E. coli.

References

Kostan, J. Sjöblom, B. et al (2010) Structural and functional characterisation of the chlorite dismutase from the nitrite-oxidizing bacterium “Candidatus Nitrospira defluvii”: Identification of a catalytically important amino acid residue. Journal of Structural Biology, Vol 172 (issue 3), pp 331 - 342 [online]

Maixner, F. Wagner, M. et al. (2008) Environmental genomics reveals a functional chlorite dismutase in the nitrite‐oxidizing bacterium ‘Candidatus Nitrospira defluvii’. Environmental Microbiology, Vol 10 (issue 11), pp 3043-3056 [online]