Parts

This year, we handed in 37 documented bio-brick parts, including all those we used in our project this year.

2.1 Basic Part: LBD of truncated GR (BBa_K4414000)

This part is a basic part, the ligand binding domain (LBD) of glucocorticoid receptor (GR). GR functions as a transcription factor that binds to glucocorticoid response elements (GREs) in the promoters of glucocorticoid-responsive genes to activate their transcription (Lu & Cidlowski, 2005). GR consists of an N-terminus binding domain (NTD), a DNA binding domain (DBD), and a ligand binding domain (LBD). As the major functional domain, the LBD domain is responsible for sensing glucocorticoid signals and translocating the protein into the nucleus (Monsalve et al., 2019). It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression (Weikum et al., 2017). Based on these functions of LBD, we used it as a sensor for a stress detection system and added tetR to complement the functions of DBD to achieve control over downstream reporter gene expression.

Figure 1. Linear domain structure of glucocorticoid receptor (GR) (Weikum et al., 2017)

2.2 Composite Part: LBD-GSG-NES-GSG-tetR (BBa_K4414044)

This part is a composite part designed to respond to changes in glucocorticoid concentration, and in turn bind to the TCE promoter to activate transcription and translation of downstream reporter genes. This part consists of a tetR DNA binding domain (BBa_K4414009), which binds to the TCE promoter(BBa_K4016011)； and a GR LBD domain (BBa_K4414000) , which is the ligand-binding domain of the glucocorticoid receptor(GR). This LBD domain can translocate the fusion protein into the nucleus upon glucocorticoid stimulation. It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression ( Weikum et al., 2017). The tetR DNA binding domain and the GR LBD are connected by GSG-NES-GSG sequences. NES is a nuclear export signal which can translocate protein from the nucleus into the cytosol.

Figure 2. Schematic representation of the LBD-GSG-NES-GSG-tetR (BBa_K4414044) ’s function on binding to the TCE promoter and activating downstream transcription