Coding

Part:BBa_K3122000:Design

Designed by: Laura Sanchez Lara   Group: iGEM19_MADRID_UCM   (2019-10-16)
Revision as of 22:01, 21 October 2019 by Franquero (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


AEGIS's lamB display system for Gram- bacteria


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 152
    Illegal AgeI site found at 464
    Illegal AgeI site found at 1229
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 467
    Illegal BsaI.rc site found at 461


Design Notes

Our group have adapted this functional display system to type IIS enzymes grammar, and called it AEGIS's lamB display system for Gram-negative bacteria. This part was designed to use the outer membrane protein lamB of E. coli K12 strain to express any tag or short sequence to the extracellular medium on its specific permissive loop. To achieve this goal, two oppositely directed BsaI restriction sites were introduced in the sequence corresponding to the lamB permissive loop, and two new ‘scars’ were designed in a way that maintains the frameshift. What is more, those scars use the lamB very nucleotides. Therefore, it does not add any new amino acid to the sequence: when introducing a tag, the assembly is completely scarless. This is an important point: although it does not comply with RFC1000 standard at first glance, it is just because its internal BsaI recognition sites, needed for the system fo to work. Apart from that, internal restriction enzymes were modified so they were deleted and codon usage was optimized for Escherichia coli.


Source

.

References