Part:BBa_K4192022

FpOAR, oxalate efflux channel protein

It can discharge oxalate form thallus, so that the bacteria can adapt to the high concentration of oxalic acid environment. It is original from Fomitopsis palustris strain TYP6137, and we have tested it in E.coli and Pseudomonas fluorescens.

Usage

Basic part FpOAR, encoding the efflux channel of oxalate.In our project, we used this part to efflux oxalic acid produced by engineering bacteria to achieve extracellular enrichment of oxalic acid. After exogenously adding calcium ions, calcium oxalate protective film was formed.

Characterization

The part comes from Fomitopsis palustris originally, therefore we need to test if the gene FpOAR can work in prokaryotes as well.

For the engineered bacteria with the FpOAR, we expected that it can have stronger viability in medium with oxalate than the control group. Bacteria can spontaneously absorb oxalate in the medium, but oxalate is harmful to bacteria because it chelates calcium ions. Consequently, engineered bacteria with the FpOAR can efflux oxalate, ends up with a better survival state. To perform this assay, we designed a gene circuit to insert FpOAR into pUC18, a commonly used vector in E. coli. As for control, we use the E. coli which has transferred an empty pUC18 vector.

We built the genetic circuit using One Step Cloning Kit II (Vazyme Biotech, China) to connect RBS sequence (BBa_K4192000) upstream to BBa_K4192022. We successfully constructed the above sequences and verified them by colony PCR and sequencing.

We tested the gene by simple quantitive test of the concentration of bacteria in the liquid medium by testing the OD595 of the bacterial solution after 12 hours of culture and induction.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 158
  Illegal PstI site found at 301
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 158
  Illegal PstI site found at 301
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 158
  Illegal BglII site found at 1161
  Illegal XhoI site found at 973
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 158
  Illegal PstI site found at 301
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 158
  Illegal PstI site found at 301
  
• 1000
  COMPATIBLE WITH RFC[1000]