Part:BBa_K1352011

Ag43+FLAG+MCS for surface display of foreign peptides

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 442
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 745
  Illegal NgoMIV site found at 1969
  Illegal NgoMIV site found at 2479
  Illegal NgoMIV site found at 2500
  Illegal AgeI site found at 1495
  Illegal AgeI site found at 2239
  Illegal AgeI site found at 2653
  Illegal AgeI site found at 2895
  
• 1000
  COMPATIBLE WITH RFC[1000]

Antigen 43 (sometimes called Ag43 or fluffing protein) is a phase-variable outer membrane protein encoded by flu gene. It is native to E.Coli K12 strain and is usually expressed at about 50, 000 copies/cell. Ag34 precursor is 1039 amino acids long and subsequently becomes cleaved into alpha and beta chains (499 and 488 amino acids long respectively). The beta subunit forms a β-barrel pore via which alpha-subunit translocates to the cell surface, and with which it remains non-covalently joined. The surface alpha chain can be released by a brief heat treatment at approx. 60oC.

Ag43 is an autotransporter protein, therefore it possesses all information necessary for translocation to the cell surface in its coding sequence.

Ag43 mediates autoaggregation, via a velcro-like mechanism (Heras et. al., 2014), and plays a role in E.coli biofilm formation.

Interestingly, the alpha subunit is able to express foreign peptide sequences on E.coli cell surface if inserted just in front of codon 148 (Kjærgaard et. al., 2002).

This BioBrick is composed of an Ag43 coding sequence with an in-frame FLAG epitope tag flanked by BglII and HindIII multiple cloning sites inserted within the alpha-subunit in front of codon 148 of Ag43.

Structure of the BioBrick was designed to allow easy insertion of foreign protein sequences of choice at codon 148 with a simple restriction digest with BglII and HindIII followed by ligation. Potential uses of this BioBrick include surface display of foreign peptide sequences and synthetic vaccines production.

Depending on the length, desired foreign peptide sequence can be purchased as oligos or a synthetic piece of DNA. In order to keep the foreign protein sequence in-frame, a following template should be used:

5' GCT GTG AGA TCT (your sequence) AAG CTT AAC ACC 3'

BglII recognition site; HindIII recognition site; sequence adjacent to foreign peptide insertion site (regular font)