Plasmid_Backbone

Part:BBa_K3394006

Designed by: Talia Shaler   Group: iGEM20_BGU-Israel   (2020-10-17)


AIDA-I auto-display system

Since cellulose cannot cross the membrane, we engineered E. coli to express the cellulolytic complex on its surface through AIDA-I auto-display system.

The AIDA-I auto-display system we chose, utilizes the translocation mechanism of passenger proteins across the outer membrane through a C-terminal beta-barrel and its preceding linking region. We planned to use JK321 E. coli strain which is a DsbA-negative mutant strain of E. coli that facilitates the autodisplay of passenger proteins and contains the AIDA-I auto display plasmid. The ompT-gene (Outer membrane protease T) in the DsbA-negative strain was mutated in order to obtain a stable surface display of the recombinant passenger proteins.


Figure 1: AIDA-I auto-display system. Structure of the polyprotein precursor and the secretion mechanism of the autotransporter proteins (SP, signal peptide; IM, inner membrane; PP, periplasm; OM, outer membrane). source: Jose, J., & Meyer, T. F. (2007). The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications. Microbiology and Molecular Biology Reviews, 71(4), 600–619.

In figure 1, we can see the structure of the polyprotein precursor. By the use of a typical signal peptide (SP), a precursor protein is transported across the inner membrane. After arrival at the periplasm, the C-terminal part of the precursor folds as a porin-like structure, a so-called beta-barrel within the outer membrane, and the passenger proteins are transmitted to the cell surface.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 241
    Illegal EcoRI site found at 937
    Illegal EcoRI site found at 4139
    Illegal PstI site found at 289
    Illegal PstI site found at 875
    Illegal PstI site found at 3387
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 241
    Illegal EcoRI site found at 937
    Illegal EcoRI site found at 4139
    Illegal PstI site found at 289
    Illegal PstI site found at 875
    Illegal PstI site found at 3387
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 241
    Illegal EcoRI site found at 937
    Illegal EcoRI site found at 4139
    Illegal BamHI site found at 1139
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 241
    Illegal EcoRI site found at 937
    Illegal EcoRI site found at 4139
    Illegal PstI site found at 289
    Illegal PstI site found at 875
    Illegal PstI site found at 3387
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 241
    Illegal EcoRI site found at 937
    Illegal EcoRI site found at 4139
    Illegal PstI site found at 289
    Illegal PstI site found at 875
    Illegal PstI site found at 3387
    Illegal NgoMIV site found at 1351
    Illegal NgoMIV site found at 1398
    Illegal NgoMIV site found at 1766
    Illegal NgoMIV site found at 1926
    Illegal NgoMIV site found at 2280
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 3213


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