Part:BBa_K1149003

pelB-pueA expression

This part expresses a plastic degradation enzyme under the control of xylose inducible promoter.

The enzyme is the pueA from Pseudomonas chlororaphis, that encodes carboxylesterase that breaks down ester bonds in polyurethane. This protein is [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00231.x/full naturally extracellular], it has a C-terminal secretion signal located downstream of a domain containing three glycine-rich repeats. These glycine-rich repeats are nonapeptide motifs involved in Ca2+-binding found in proteins secreted via type I systems. It uses the RTX system. To ensure secretion in E.coli, we have also added a pelB seretion tag to the N-terminal. We also added a 6xHistidine tag for Western blotting and protein purification purposes.

Theoretical pathway of PUR degradation by esterase:

references:

[http://www.selu.edu/acad_research/depts/biol/faculty/publications/pdf/2012/howard2012.pdf Howard, Gary 2012 pg:371 -3, Polyurethane Biodegradation, Environmental Science] [RTX proteins: a highly diverse family secreted by a common mechanism http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00231.x/full] Stern, R. V., and G. T. Howard. 2000. The polyester polyurethane gene (pueA) from Pseudomonas clororaphis encodes lipase. FEMS Microbiol. Lett. 185:163–168.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 99
  Illegal BglII site found at 964
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]