TY - UNPB
T1 - Profiling bacterial kinase activity using a genetic circuit
AU - van der Helm, Eric
AU - Bech, Rasmus
AU - Lehning, Christina Eva
AU - Vazquez-Uribe, Ruben
AU - Sommer, Morten Otto Alexander
PY - 2017
Y1 - 2017
N2 - Phosphorylation is a post-translational modification that regulates the activity of several key proteins in bacteria and eukaryotes. Accordingly, a variety of tools has been developed to measure kinase activity. To couple phosphorylation to an in vivo fluorescent readout we used the Bacillus subtilis kinase PtkA, transmembrane activator TkmA and the repressor FatR to construct a genetic circuit in E. coli. By tuning the repressor and kinase expression level at the same time, we were able to show a 4.2-fold increase in signal upon kinase induction. We furthermore validated that the previously reported FatR Y45E mutation1 attenuates operator repression. This genetic circuit provides a starting point for computational protein design and a metagenomic library-screening tool.
AB - Phosphorylation is a post-translational modification that regulates the activity of several key proteins in bacteria and eukaryotes. Accordingly, a variety of tools has been developed to measure kinase activity. To couple phosphorylation to an in vivo fluorescent readout we used the Bacillus subtilis kinase PtkA, transmembrane activator TkmA and the repressor FatR to construct a genetic circuit in E. coli. By tuning the repressor and kinase expression level at the same time, we were able to show a 4.2-fold increase in signal upon kinase induction. We furthermore validated that the previously reported FatR Y45E mutation1 attenuates operator repression. This genetic circuit provides a starting point for computational protein design and a metagenomic library-screening tool.
U2 - 10.17605/OSF.IO/AKG5X
DO - 10.17605/OSF.IO/AKG5X
M3 - Working paper
BT - Profiling bacterial kinase activity using a genetic circuit
PB - Novo Nordisk Foundation Center for Biosustainability
ER -