Volume 16 Supplement 1

Abstracts from the 7th International Conference on cGMP Generators, Effectors and Therapeutic Implications

Open Access

Design and directed evolution of genetically encoded cGMP sensors

BMC Pharmacology and Toxicology201516(Suppl 1):A48

https://doi.org/10.1186/2050-6511-16-S1-A48

Published: 2 September 2015

Imaging cGMP transients in in vivo models is very challenging and therefore requires probes that are highly optimized for this task. Several key factors are to be considered while developing such a biosensor: affinity and dynamic range – to cover physiologically relevant concentration ranges, sensitivity – to facilitate meaningful imaging of small variations in concentration, and overall brightness and stability – to increase signal/noise ratio and provide comparable results through a variety of compartments and tissues.

We seek to generate a robust genetically encoded FRET-sensor for cGMP imaging. Our approach adapts protocols that our lab developed and has already successfully applied in the development of Ca2+ biosensors [1, 2]. This method combines an initial rational design approach with several iterations of directed evolution. Rational design of these probes supplies prototypes, which are turned into partially randomized libraries. These libraries are subsequently screened in E.coli by means of a semiautomatic high throughput process. Promising candidates are biochemically characterized in vitro and later on tested in cell culture.

Initial results already show substantial improvements over existing cGMP biosensors and indicate, that the success in the field of Ca2+ imaging can be repeated for cGMP.

Authors’ Affiliations

(1)
Max Planck Institute of Neurobiology

References

  1. Thestrup T, Litzlbauer J, Bartholomaus I, Mues M, Russo L, Dana H, et al: Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes. Nat Methods. 2014, 11 (2): 175-182. 10.1038/nmeth.2773.View ArticlePubMedGoogle Scholar
  2. Litzlbauer J, Schifferer M, Ng D, Fabritius A, Thestrup T, Griesbeck O: Large Scale Bacterial Colony Screening of Diversified FRET Biosensors. PLoS One. 2015, 10 (6): e0119860-10.1371/journal.pone.0119860.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© Fabritius and Griesbeck et al. 2015

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Advertisement