Difference between revisions of "Part:BBa K633002"

 
 
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<partinfo>BBa_K633002 short</partinfo>
 
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We used Rybosome Binding Site BBa_B0034 designed by by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr, evaluated by Warsaw's 2010 team. we added membrane protein signal peptide phoA and cellulase immediately next to it
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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).
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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 with 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:
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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.
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Richeter, S.; Brüser, T. (2005). Targeting of unfolding PhoA to the TAT Translocon of Escherichia coli. Journal of Biological Chemistry 280 (52), 42723-42730.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 00:01, 20 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 with 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]