Automation of E.coli cell based assay for CRE1/AHK4 receptor characterization


Author(s) and Affiliations

Klimeš P.^1,2, Turek D.^1, Mazura P.^1, Spíchal L.^3, Brzobohatý B.^1

^1Laboratory of Plant Molecular Biology, Institute of Biophysics, Academy of Sciences of the Czech
Republic, v.v.i. and CEITEC-Central European Institute of Technology,
Mendel University in Brno, Brno, Czech Republic

^2Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic

^3University & Institute of Experimental Botany ASCR & Centre of the Region Haná, Olomouc, Czech
Republic


Abstract (200 words or less)

Histidine kinase CRE1/AHK4 is a plant hormone receptor providing cytokinin signalling across the
membrane structures in the plant cell. Functional studies of histidine kinases are crucial for
understanding many signal processes in plants as well as in prokaryotes where these signalling
systems dominate.  Possibility of expression of CRE1/AHK4 receptor in E. coli allows studying
responses of this receptor to various potential ligands. Artificial signalling pathway in E. coli
depending on the receptor function leads to expression of reporter gene encoding enzyme
beta-galactosidase. Beta-galactosidase is able to hydrolyse an artificial substrate to form a
measurable product. The amount of formed product is proportional to the amount of added hormone. We
used liquid handling robotics to automate and miniaturise an existing method [1] for response
measurement. Advantages of the method are lower consumption of all solutions and fast, walk away
setup. The performance of the method was examined and compared with current state of the art
solution. Using the improved method we studied response of the CRE1/AHK4 receptor with a several
ligands in the 384-well microtitration plate. We confirmed usability of the method and obtained new
data for further receptor functional studies.


[1] Spíchal L., et al. (2004): Two Cytokinin Receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3,
Differ in their Ligand Specificity in a Bacterial Assay, Plant Cell Physiol. 45(9): 1299–1305