Part:BBa_K2695008

N. defluvii signal peptide for chlorite dismutase

Usage and Biology

Although Nitrospira defluvii is a nitrite-oxidizing bacterium, surprisingly a highly active chlorite dismutase was recently identified (Maixner et. al, 2008). This enzyme is located in the periplasm of the bacterium.

As our project involves expressing these enzymes in E. coli we needed to determine whether the signal peptides from the nitrate-oxidizing bacteria are able to export the enzymes into the periplasm. We therefore inserted the signal peptide from N. defluvii Cld at the N-terminal of superfolded GFP and using the T7 promoter expressed the resulting protein in BL21(DE3).

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 119
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 9
  Illegal BsaI.rc site found at 823
  

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
• 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-GFP-tag primary antibody raised in mouse and and anti-mouse secondary antibody raised in goat conjugated to alkaline phosphatase. Cell fractionation was performed to separate the cytoplasmic and periplasmic fractions and allow us to perform a Western blot in these specific areas of the cell.

References

Maixner, F., Wagner, M., Lucker, S., Pelletier, E., Schmitz-Esser, S., Hace, K., Spieck, E., Konrat, R., Le Paslier, D., Daims, H., 2008. Environmental genomics reveals a functional chlorite dismutase in the nitrite-oxidizing bacterium ‘Candidatus Nitrospira defluvii’. Environ. Microbiol. 10, 3043–3056.