"
Team:Brown/Parts
From 2009.igem.org
Team 3 Parts
Tar-EnvZ
In our system, the Tar-EnvZ (or Taz) chimera protein is used to indicate and signal the presence of a chemical ligand. Endogenous to E. coli cells, Tar has three domains: a periplasmic ligand binding receptor domain, a transmembrane domain, and an intracellular kinase domain. When aspartate binds to the Tar receptor domain, the kinase domain subsequently propagates a message by modifying intracellular components, ultimately resulting in regulation of flagella rotation. Also endogenous to E. Coli is the EnvZ protein, an inner membrane kinase which responds to changes in osmolarity. When activated, the EnvZ kinase phosphorylates transcription factor OmpR, which subsequently activates transcription of the OmpC gene. Tar-EnvZ (Taz) is a chimera protein, manufactured by Inouye, et al. We obtained the gene from his lab and Biobricked it. Taz comprises of the aspartate chemoreceptor region of Tar, the transmembrane region of Tar, and the intracellular kinase region of EnvZ. The genes were fused by digesting both with NdeI and ligating the overlapping ends together. The cut site is between amino acids H256 and M257.
[[1]]|DNA Sequence|
OmpC promoter-RFP
This construct is a reporter for EnvZ activation. Once activated, EnvZ phosphorylates transcription factor OmpR, which in turn activates transcription of the OmpC gene. This cassette contains the promoter region of OmpC (BBa_R0082), placed over a red fluorescence protein gene (BBa_E1010). The ribosome binding site is . There is also a double terminator sequence. [[2]]|DNA Sequence|
