Difference between revisions of "Part:BBa K633002"
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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).
 
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).
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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).
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Since the objective of our project is to display chimeric proteins in the cell surface of ''Escherichia coli'', a signal peptide is needed to translocate the protein into the periplasm. The signal sequence of phoA can be used to send bacterial periplasmic proteins across the membrane. Becker et al. (2005) also reported the efficient translocation of EstA through the ''E. Coli'' cytoplasmic membrane by adding to the native EstA sequence phoA signal peptide sequence. It is expected then that CelD will be secreted into the extracellular medium using this part alone, or be displayed on the microbial cell surface, when fused with EstA membrane protein (BBa_K63001).
 
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'''References:'''
 
'''References:'''

Revision as of 21:54, 19 October 2011

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 is to display chimeric proteins in the cell surface of Escherichia coli, a signal peptide is needed to translocate the protein into the periplasm. The signal sequence of phoA can be used to send bacterial periplasmic proteins across the membrane. Becker et al. (2005) also reported the efficient translocation of EstA through the E. Coli cytoplasmic membrane by adding to the native EstA sequence phoA signal peptide sequence. It is expected then that CelD will be secreted 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:



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]