Part:BBa_K5091007:Experience

Engineering and Designing FAB

Building on the 2023 Lethbridge Collegiate Teams work, which aimed to develop and validate the components for a protein-based detection system, our project, C.R.O.P.S., advanced that goal by focusing on the design and engineering of a fragment antigen-binding (FAB) antibody. This antibody was conceptualized through binding and molecular dynamics simulations to optimize the interactions with the target protein PbEL04 as a way to effectively detect the clubroot pathogen.

Transformation of FAB Construct

The transformation of the FAB construct into Rosetta and DH5α cells is a crucial step in biotechnology for recombinant protein expression. The process begins with the introduction of plasmids containing the genetic sequences encoding the FAB using heat shock into competent Rosetta or DH5α cells. Both cell lines were selected for our plasmid transformation as they provide different benefits for our protein production. The FAB construct was successfully transformed into both cell lines.

pET28a plasmids were cloned to contain FAB constructs. The plasmids were transformed into Rosetta and DH5α cells. Rosetta (DE3) electrocompetent cells were chosen as they possess additional plasmids that enhance the expression of proteins containing rare codons, which is often necessary for eukaryotic-derived proteins, like the FAB. They are the cell line that is used in the protein overexpression and purification. In contrast, DH5α cells were selected for their high transformation efficiency and ability to maintain plasmid stability, making them ideal for cloning. DH5α cells were used in any plasmid preparation, as they produced a large amount of plasmids containing the protein coding regions.

The plasmid coding for FAB was transformed into Rosetta and DH5α cells.

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