This part consists of a chez coding region followed by a 6-Histidine tail
CheZ coding sequence (Chemotaxis protein)
CheZ is one of the 6 Che proteins used in chemeotaxis. CheZ is a phosphatase which dephosphorylates CheY which in turn then allows the cell to run and tumble, wheres if CheZ is absence, CheY is phosphorylated, and the cell tumbles exclusively. (Topp and Gallivan J. Am. Chem. Soc. 129, p6865-6873)
EnvZ* is a transmenbrane kinase that phosphorylates ompR the natural inhibitor of pFlhDC independently of any outer signal.
bacterial genome of mutated (I281S) strain for EnvZ.
This part promotes the constitutive expression of the curli cluster (csgD), proteins involved in cell-cell adhesion.
This part regulates curli expression within the cell via transcriptional activation. The construct can be repressed in the presence of the TetR repressor.
cheZ, chemotaxis regulator, protein phosphatase for CheY
Polar localization is dependent on CheA-short. CheZ is a cytosolic phosphatase which functions in the chemotaxis signal transduction complex. Plays an important role in bacterial chemotaxis signal transduction pathway by accelerating the dephosphorylation of phosphorylated CheY (CheY-P).
how to control the proper expression of cheZ
CheZ: phosphatase which dephosphorylates CheY-P and thus plays an important role in E.Coli chemotaxi
This part encodes CheZ, which is a phosphatase which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise. E.coli lacking the cheZ gene cannot dephosphorylate CheY-P, tumble incessantly, and are thus nonmotile while inducing the expression of CheZ can restore motility in a CheZ knockout strain by reactivating wild-type chemotaxis.
We get it from the genome of Top10 by PCR.
cheZ ligated to a weak promoter. This part express CheZ protein weakly and will induce weak mobility in delta cheZ E.coli strain.
There is a point mutation at position 56 of CheZ coding sequence with have little effect to its function and the bases corresponding to stop codon is changed to bases coding a more frequently used stop codon.
The promoter used here is from Partsregistry. The CheZ gene is from E.coli strain MG1655 by colony PCR and aligned with CheZ sequence at EcoGene database.
Fusion protein Trg-EnvZ for signal transduction
We created a hybrid gene coding for a protein in which Trg (chemoreceptor) contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ (osmosensor) contributed its cytoplasmic kinase/phosphatase domain.
Characterize efficiency of RBS with chemotaxis
BBa_R0010 BBa_B0033 BBa_K629003 BBa_B0015 Plac-RBS(0.01)-CheZ-TT This part can express CheZ protein which decide the E.coli whether tumble of swim straight.In return, we can charecterize the efficiency of RBS and Promoter by measure the distance E.coli migration. 3H:13-P3-3H BBa_R0010 2L:14-P2-2L BBa_B0033 18G:14-P1-18G BBa_K629003 4F:13-P3-4F BBa_B0015
1.link RBS and Promoter is too short to link to other gene part.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Coding sequence for Tar-EnvZ fusion protein. Tar is the bacterial chemotaxis receptor for aspartate. EnvZ is an osmosensor kinase, which, through phosphorylation of OmpR, regulates transcription of porin genes. This fusion protein combines the extracellular and transmembrane domains of Tar with the intracellular domain of EnvZ. The fusion converts chemical binding of aspartate to production of transcription factors.
A silent point mutation was made at base 423 (A -> C) to eliminate an XbaI restriction enzyme site. The mutation was designed to match codons with similar frequencies of usage in E. coli.
This is the coding sequence of ''glgC'' (ADP-glucose pyrophosphorylase) from ''Escherichia coli'' JM109 with the substitution G336D. This mutation is known to cause increased activity of ADP-glucose pyrophosphorylase in the absence of the activator fructose 1,6-bisphosphate (FBP), high affinity for FBP and substrates lower affinity for the inhibitor AMP. (Leung ''et al'', 1986; Meyer ''et al.'', 1988)
The mutation G336D was achieved by the substitution of A for G at position 1076 (codon GGC->GAC) by MABEL.
pck codes for Phosphoenolpyruvate carboxykinase (Pck), a kinase involved in gluconeogenesis in E. coli
No codons were modified to make this part
This part contains the cheZ operon coding region without an rbs nor a terminator. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement.
This part is without promoter, rbs or terminator.
The origin of the cheZ gene comes from E. Coli strain MG1655 chromosome.
FF_FF ansB promoter-rbs-cheZ_histag-terminators
This part consist of a chez protein linked to a double terminator. The cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement. The chez coding region has a 6-Histidine tail and do not contain any rbs.
The chez-histag coding region is self-designed The double terminator is from the registry
Synthetic periplasmic binding protein that docks a vanillin molecule
glgC coding sequence encoding ADP-glucose pyrophosphorylase
This is the coding sequence of ''glgC'' from ''Escherichia coli'' JM109. It encodes ADP-glucose pyrophosphorylase, which catalyses Glucose 1-phosphate + ATP -> ADP-glucose + PPi, a step in the production of glycogen from glucose.
The genomic DNA sequence contains to EcoRI sites, both of which have been mutated out using the mutagenesis by blunt-ended ligation (MABEL) technique
The cheZ motility protein is a protein kinase which dephosphorylates cheY. cheY regulates the clockwise and counterclockwise rotation of the motor flagella. If cheY is phosphorylated the motor flagella are not in sync and the bacterium tumbles. If cheY is not phosphorylated the motor flagella are in sync and the bacterium runs (often towards a chemoattractant)
This part codes for the protein LsrK which phosphorylates AI-2 which acts to promote the lsrA promoter.
This sequence is found in the E. coli strain MG1655.