Part:BBa_K4130003

IgG F(c) Binding Protein, EibA

Biology

E. coli Ig-binding proteins (Eibs) are a wide-spread class of proteins within intimin-negative, shiga-toxin producing enterohemorrhagic E. coli (EHEC) strains [1]. First discovered in strain ECOR-9 [2, 3], the class has grown to include EibA, C, D, E, F, and G. The class is united in its common characteristic of immunoglobulin (Ig) binding activity.

Immunoglobulins, also known as antibodies, are a family of heterodimeric proteins produced by B cells capable of binding with great specificity to respective antigens. Igs are typically composed of a “variable region”, responsible for binding of the antigen, and a “constant region” with no affinity for the antigen. Igs are further classified according to their “constant regions”, into 5 classes: IgM, IgG, IgA, IgD, and IgE. [4]

E. coli Ig-binding proteins have been shown to be capable of binding the constant region of assorted Ig classes. Affinity and specificity for different classes of Ig proteins varies between Eib family members, with dissociation constants on the 100nM scale [5].

EibA (from E. coli strain ECOR-9) is a 392 amino acid, >120kDa [3] member of the Eib family with high affinity for the protein IgG (Kd = 209nM) [5]. The exact role of EibA in bacteria is unknown, though related proteins such as YadA are implicated in virulence pathways [6].

Usage

Given the Ig-binding properties of EibA, we chose to exploit it for whole-cell agglutination/aggregation assays to detect small molecules and proteins. By incubating EibA-expressing bacteria with immunoglobulin antibodies, we hoped to be able to detect various antigens. Addition of the antigen would lead to bivalent binding interactions, causing agglutination/aggregation of the cells. Agglutination/aggregation can be observed visually or quantitatively using a spectrophotometer.

Uniquely, this strategy of detection has the potential to be applied to nearly any antigen with zero need for further genetic engineering. Previous whole-cell bacterial sensors have been focused on expressing antibodies or antibody fragments on the surface of the bacterium, meaning that each strain is specific to an antigen. By expressing a general antibody-binding protein, a single strain can be utilized for the detection of multiple antigens.

Design

We therefore designed a genetic circuit composed of the EibA gene from E. coli strain ECOR-9, a rhamnose-inducible promoter (BBa_K914003), strong ribosome binding site (BBa_B0034), and double-terminator (BBa_B0015). Synthesized parts were assembled into the chloramphenicol-resistance conferring pSB1C3 vector.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]