TY - JOUR
T1 - Light-controlled gene expression in yeast using photocaged Cu2+
AU - Kusen, Peter M.
AU - Wandrey, Georg
AU - Krewald, Vera
AU - Holz, Martina
AU - Berstenhorst, Sonja Meyer zu
AU - Büchs, Jochen
AU - Pietruszka, Jörg
PY - 2017/9/20
Y1 - 2017/9/20
N2 - The manipulation of cellular function, such as the regulation of gene expression, is of great interest to many biotechnological applications and often achieved by the addition of small effector molecules. By combining effector molecules with photolabile protecting groups that mask their biological activity until they are activated by light, precise, yet minimally invasive, photocontrol is enabled. However, applications of this trendsetting technology are limited by the small number of established caged compound-based expression systems. Supported by computational chemistry, we used the versatile photolabile chelator DMNP-EDTA, long-established in neurobiology for photolytic Ca2+ release, to control Cu2+ release upon specific UV-A irradiation. This permits light-mediated control over the widely used Cu2+-inducible pCUP1 promoter from S. cerevisiae and thus constitutes the first example of a caged metal ion to regulate recombinant gene expression. We screened our novel DMNP-EDTA-Cu system for best induction time and expression level of eYFP with a high-throughput online monitoring system equipped with an LED array for individual illumination of every single well. Thereby, we realized a minimally invasive, easy-to-control, parallel and automated optical expression regulation via caged Cu2+ allowing temporal and quantitative control as a beneficial alternative to conventional induction via pipetting CuCl2 as effector molecule.
AB - The manipulation of cellular function, such as the regulation of gene expression, is of great interest to many biotechnological applications and often achieved by the addition of small effector molecules. By combining effector molecules with photolabile protecting groups that mask their biological activity until they are activated by light, precise, yet minimally invasive, photocontrol is enabled. However, applications of this trendsetting technology are limited by the small number of established caged compound-based expression systems. Supported by computational chemistry, we used the versatile photolabile chelator DMNP-EDTA, long-established in neurobiology for photolytic Ca2+ release, to control Cu2+ release upon specific UV-A irradiation. This permits light-mediated control over the widely used Cu2+-inducible pCUP1 promoter from S. cerevisiae and thus constitutes the first example of a caged metal ion to regulate recombinant gene expression. We screened our novel DMNP-EDTA-Cu system for best induction time and expression level of eYFP with a high-throughput online monitoring system equipped with an LED array for individual illumination of every single well. Thereby, we realized a minimally invasive, easy-to-control, parallel and automated optical expression regulation via caged Cu2+ allowing temporal and quantitative control as a beneficial alternative to conventional induction via pipetting CuCl2 as effector molecule.
KW - BioLector
KW - Caged compound
KW - DMNP-EDTA
KW - Light-regulated expression
KW - Optogenetics
KW - pCUP1
UR - http://www.scopus.com/inward/record.url?scp=85018999504&partnerID=8YFLogxK
UR - http://dx.doi.org/10.1016/j.jbiotec.2017.04.032
U2 - 10.1016/j.jbiotec.2017.04.032
DO - 10.1016/j.jbiotec.2017.04.032
M3 - Article
AN - SCOPUS:85018999504
SN - 0168-1656
VL - 258
SP - 117
EP - 125
JO - Journal of Biotechnology
JF - Journal of Biotechnology
ER -