Primer-Aided Truncation for the Creation of Hybrid Proteins (PATCHY)Learn less
Many proteins consist of individual domains that are connected by so-called linker sequences with widely differing length, sequence and structure. In signal receptors, linkers connect the sensor unit that perceives cognate signals to the effector unit which elicits physiological responses. Signal transduction from sensor to effector hinges on the intervening linker; modifications in the linker as small as the exchange, the insertion or removal of a single amino acid residue can profoundly alter receptor output. For the design of signal receptors, this poses a particular challenge and often entails the construction and testing of multiple linker variants. To aid this process, we have developed the PATCHY method (Primer-Aided Truncation for the Creation of Hybrid Proteins) for the facile construction of defined gene libraries in which individual members differ in length and sequence of the linker between two gene fragments A and B.
PATCHY is based on the amplification of a precursor plasmid with staggered primers as illustrated in the figure. We have developed a Python script which designs the required primers for PCR amplification. For details, cf. below references. To obtain the program, click on [Download].
- Ohlendorf, R., Schumacher, C.H., Richter, F., and Möglich, A. (2016). Library-Aided Probing of Linker Determinants in Hybrid Photoreceptors. ACS Synth. Biol. 5, 1117-1126. [Pubmed]
- Stabel, R., Stüven, B., Ohlendorf, R., and Möglich, A. (2017). Primer-Aided Truncation for the Creation of Hybrid Proteins. Meth. Mol. Biol. 1596, 287-304. [Pubmed]
Upgrading a Microplate Reader for Photobiology and All-optical ExperimentsLearn less
Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. The setup is easily assembled and thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.
For details, cf. below reference. To obtain the program, click on [Download].
- Richter, F., Scheib, U.S., Mehlhorn, J., Schubert, R., Wietek, J., Gernetzki, O., Hegemann, P., Mathes, T., and Möglich, A. (2015). Upgrading a microplate reader for photobiology and all-optical experiments. Photochem. Photobiol. Sci. 14, 270-279. [Pubmed]
Programmable LED Arrays for Photobiological ExperimentsLearn less
Experiments in photobiology frequently demand the illumination of samples with light of specific wavelength and intensity for defined times. To facilitate such experiments, we have developed an Arduino-based, programmable LED array for the illumination of microtiter plates. Using a graphical user interface the color and intensity of an array of 8x8 LEDs can be configured.
The original LED array (above) used a commercially available LED matrix with emission in the blue, green and red regions of the visible spectrum. To enable experiments with photoreceptors responding to other light colors, we redesigned the setup to allow use of custom LEDs.
- Hennemann, J., Iwasaki, R.S., Grund, T.N., Diensthuber, R.P., Richter, F., and Möglich, A. (2018). Optogenetic Control by Pulsed Illumination. Chembiochem 19(12), 1296-1304. [Pubmed]
- Stüven, B., Stabel, R., Ohlendorf, R., Beck, J., Schubert, R., and Möglich, A. (2019). Characterization and engineering of photoactivated adenylyl cyclases. Biol Chem in press [doi]