Part:BBa_K3122000:Design
AEGIS's lamB display system for Gram- bacteria
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 152
Illegal AgeI site found at 464
Illegal AgeI site found at 1229 - 1000INCOMPATIBLE 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
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