A New Rapid and Quantitative Assay to Determine the Phytase Activity of Feed.

The fortification of animal feed with enzymes in order to optimize feed utilization has become a standard for the meat production industry. A method for measuring levels of active enzymes that can be carried out quickly would ensure that feed has been supplemented with the appropriate amount of enzyme. Phytase is the most widely used feed enzyme and is routinely quantified with an activity assay in a limited number of specialized laboratories. As an alternative, we report here the development of a rapid and easy method to perform a quantitative assay for the phytase from Citrobacter braakii. The method is suitable for use at local sites with a minimum lab setup and will reduce delays and potential interferences due to improper sample storage and shipment. The new assay is based on a lateral flow immunoassay that utilizes magnetic immune-chromatographic test (MICT) technology to quantify the phytase content of a feed extract. After extraction of the phytase from the feed, the sample is simply diluted and added to a reaction tube containing a specific anti-phytase antibody coupled to superparamagnetic particles. The mixture is then applied on an assay cassette, where the formed particle-antibody-phytase complexes are captured by immobilized antibodies on a nitro-cellulose strip housed in a cassette. The cassette is placed in the MICT reader that measures the magnetic signal of the captured particles. Using the calibration information stored in the cassette barcode, the signal is converted to a phytase concentration, given as phytase activity (FYT) per kilogram of feed. The accuracy and robustness of the assay compared to the ISO phytase activity assay were demonstrated through a large validation study including real feed samples from different compositions and origins. The MICT assay is the first quantitative assay for feed enzymes that is fast, reliable, and simple to use outside of a specialized reference laboratory and that is suitable for use in place of the current ISO assay.

Green Light-Controlled Gene Switch for Mammalian and Plant Cells.

The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.

Flow-based regenerable chemiluminescence receptor assay for the detection of tetracyclines.

For the first time, a flow-based regenerable chemiluminescence receptor assay is established that is eminently suited as screening method for the detection of widely used tetracyclines (TCs) in environmental and food samples. The complex functionality and high reactivity of TCs complicate the creation of immunogens which is currently the bottleneck for developing sensitive immunoassays. In this case, competitive bioreceptor assays for the analysis of small organic molecules are preferable and, moreover, flow-based regenerable bioassays are optimally suited for automated analysis applications. Therefore, the solution for rapid and sensitive analysis of TCs is the regenerable CL receptor assay with a covalently immobilized DNA oligonucleotide containing the specific operator sequence tetO to which the repressor protein TetR binds only in the absence of TCs. The TC measurements are performed on the CL microarray analysis platform MCR 3 within 30 min per sample. The LoD in spiked tap water was determined to be 0.1 µg L-1, and for 1 µg L-1 TET, recoveries of 77% ± 16% were obtained. Due to the stability of the immobilized DNA oligonucleotide and the resulting regenerability of the assay for various measurements, the new method is highly cost- and resource-efficient and ideally suited for the monitoring of environmental samples in the field. Graphical abstract.

Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors.

Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.