Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Martyna Sroka, Ana-Maria Cujba
Date created: 2014-10-02 11:00:00
Date modified: 2015-05-08 01:10:02
Ag43+FLAG+MCS(-)beta-hairpins under pBAD promoter
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K1352002_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).
The shape of α-subunit of Ag43 resembles letter L. It consists of a 'β-helix domain [which form] the stem of the letter L, followed by three rungs flanked by four β-hairpin motifs that bend the protein by about 110?? and a C-terminal (...) parallel β-helix domain [which forms] the bottom of the letter L' (Heras et. al., 2014). It has been indicated that this unique shape plays a crucial role in cell-to-cell aggregation via velcro-like mechanism, in which α-subunits form a dimer by coling around each other. This interaction is strengthened by Van der Waals interactions, hydrogen bonds and salt bridges facilitated by the L-shape (Heras et. al., 2014). Recent research demonstrates that disruption of the bend and straightening of the shape by removal of two β-hairpin sequences eliminates self-association of Ag43 proteins (Heras et. al., 2014). Removal of β-hairpins does not interfere with protein translocation to the cell surface membrane.
This BioBrick is a modified version of the Bba_K1352001 BioBrick. 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. Two beta hairpin sequences within Ag43 were removed via In-Fusion (Clonetech). Expression of Ag43 is under the control of the pBAD promoter; it includes a ribosome binding site as well as two transcriptional terminators.
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 when aggregation is undesirable, as well as synthetic vaccines production.
Notes
Maintaining ORF integritySource
PCR amplified from Bba_K1352001; two hairpin sequences (H1 268 AATVTGTNRLGAFSVVA 284; H2 341 GAAVSGTRSDGKAFSIG 357) were removed via In-Fusion (Clonetech).| Access | Instance | Definition |
| public public public public public | BBa_I0500 BBa_B0034 BBa_K1352012 BBa_B0010 BBa_B0012 | pBad/araC BBa_B0034 BBa_K1352012 BBa_B0010 BBa_B0012 |