Team:Hamburg/Improve
Improve a previous Part or Project
BioBrick BBa_C0082 of Antiquity 2004
Last year, the iGEM Team Hamburg (Finding Chlamydori) worked on and modifying the receptor tar-envZ. This receptor is registered in the iGEM data bank as a fusion protein of the tar-domain and the intracellular EnvZ-domain, although the receptor has an aspartate-binding domain consisting of 484 amino acids. 255 amino acids belong to the N-terminal Tar-domain and 229 amino acids to the C-terminal EnvZ-domain. The assembled construct is 1516 base pairs long and registered as BBa_C0082. from the iGEM Team Antiquity.
The function of both domains has already been thoroughly investigated Utsumi et al. [1] und Mise et al. [2], thereby only the periplasmic aspartate-binding domain (amino acids 26 to 193) has been expressed and studied. In the original paper by Mise et al. [2], the Tar-domain was amplified out of the genome of Escherichia coli DH5a with primer pair 5‘ – ATG GCT AGC GAT GAC GAC GAC AAG GGC AGC CTG TTT TTT TCT TC – 3‘ (forward) and 5‘ – CTC GAA TTC TCA TTA TTG CCA CTG GGC AAA TC – 3‘ (reverse), and cloned in the expression vector pET-28a(+). In addition to the N-terminal start methionine, the vector has a thrombine cleavage site upstream of the C-terminal his-tag. According to the paper, tar domain was purified using the His-tag. His-tag renders the protein aggregation-prone, thus it has been removed afterwards through the thrombine cleavage site. The non-tagged protein was used for a crystallography.
BioBrick BBa_C0082 of Antiquity 2004
Last year, the iGEM Team Hamburg (Finding Chlamydori) worked on and modifying the receptor tar-envZ. This receptor is registered in the iGEM data bank as a fusion protein of the tar-domain and the intracellular EnvZ-domain, although the receptor has an aspartate-binding domain consisting of 484 amino acids. 255 amino acids belong to the N-terminal Tar-domain and 229 amino acids to the C-terminal EnvZ-domain. The assembled construct is 1516 base pairs long and registered as BBa_C0082. from the iGEM Team Antiquity.
The function of both domains has already been thoroughly investigated Utsumi et al. [1] und Mise et al. [2], thereby only the periplasmic aspartate-binding domain (amino acids 26 to 193) has been expressed and studied. In the original paper by Mise et al. [2], the Tar-domain was amplified out of the genome of Escherichia coli DH5a with primer pair 5‘ – ATG GCT AGC GAT GAC GAC GAC AAG GGC AGC CTG TTT TTT TCT TC – 3‘ (forward) and 5‘ – CTC GAA TTC TCA TTA TTG CCA CTG GGC AAA TC – 3‘ (reverse), and cloned in the expression vector pET-28a(+). In addition to the N-terminal start methionine, the vector has a thrombine cleavage site upstream of the C-terminal his-tag. According to the paper, tar domain was purified using the His-tag. His-tag renders the protein aggregation-prone, thus it has been removed afterwards through the thrombine cleavage site. The non-tagged protein was used for a crystallography.
