Part:BBa_K633002

RBS+signal peptide phoA+Cellulase

This part consists of a ribosome binding site (BBa_0034), along with a gene segment encoding the leader sequence of alkaline phosphatase, phoA, and the cellulase CelD (BBa_K63000). Since the objective of our project was to display chimeric proteins in the cell surface of Escherichia coli, a signal peptide was needed to translocate the protein into the periplasm. Alkaline phosphatase PhoA normally cannot fold properly in the cytoplasm due to a necessity of disulfide formation (Richeter et al., 2005), so the signal sequence phoA can be used to send bacterial periplasmic proteins across the membrane. Becker et al. (2005) reported that an efficient translocation of the protein membrane EstA through the E. Coli cytoplasmic membrane was achieved by replacing the native EstA sequence with phoA. It is expected then that CelD will be excreted into the extracellular medium using this part alone, or be displayed on the microbial cell surface, when fused with EstA membrane protein (BBa_K63001).

References:

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Becker, S.; Theile, S.; Heppeler, N.; Michalczyk, A.; Wentzel, A.; Wilhelm, S.; Jaeger, K.; Kolmar, H. (2005). A generic system for the Escherichia coli cell-surface display of lipolytic enzymes. FEBS letters 579, 1177-1182.
Richeter, S.; Brüser, T. (2005). Targeting of unfolding PhoA to the TAT Translocon of Escherichia coli. Journal of Biological Chemistry 280 (52), 42723-42730. Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1908
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 721
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 585
  Illegal AgeI site found at 228
  Illegal AgeI site found at 644
  Illegal AgeI site found at 1473
  
• 1000
  COMPATIBLE WITH RFC[1000]