Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Martyna Sroka, Ana-Maria Cujba
Date created: 2014-10-01 11:00:00
Date modified: 2015-05-08 01:10:02
Ag43+FLAG+MCS for surface display of foreign peptides
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K1352011_sequence (Version 1) |
Description
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 (Kj??rgaard et. al., 2002).
This BioBrick was designed for E.coli surface expression of foreign peptides. It 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.
This basic part is a part of the composite BioBrick Bba_K1352001.
Notes
FLAG epitope tag flanked by BglII and HindIII multiple cloning sites was designed to be in frame of the Ag43 ORF; it is optimized for E.coli codon bias.Source
This part was PCR amplified from BioBrick Bba_K1352000 and subjected to In-Fusion (Clonetech) to insert in-frame FLAG+MCS| Sequence Annotation | Location | Component / Role(s) |
| Ag43+FLAG+MCS BglII-FLAG-HindIII | 1,3156 442,477 | feature/cds CDS sequence_feature feature/misc |