Part:BBa_K2695003

A. suillum Signal Peptide for Perchlorate Reductase (Subunit A)

Usage and Biology

Perchlorate reducing bacteria reduce the substrate perchlorate all the way to oxygen and chloride, via a toxic intermediate chlorite. Perchlorate-associated oxidative stress, the toxicity of chlorite, and the potential of producing reactive chlorine species are likely reasons why perchlorate reduction occurs in the periplasm of these bacteria. Signal peptides are at the N-terminal of the majority of the enzymes involved in this pathway to enable export from the cytoplasm into the periplasm.

As our project involves expressing these enzymes in E. coli we needed to determine whether the signal peptides from the perchlorate reducing bacteria are able to export the enzymes into the periplasm. We therefore inserted the signal peptide from A. suillum PcrA (an enzyme from the pcrABCD operon) at the N-terminal of superfolder 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 124
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 8
  Illegal BsaI.rc site found at 828
  

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.