Yeast biopanning against site-specific phosphorylations in tau.

The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer's Disease and other neurodegenerative diseases. However, there is a lack of phospho-specific monoclonal antibodies and limited validation of their binding specificity. Here, we report a novel approach using yeast biopanning against synthetic peptides containing site-specific phosphorylations. Using yeast cells displaying a previously validated phospho-tau (p-tau) single-chain variable region fragment (scFv), we show selective yeast cell binding based on single amino acid phosphorylation on the antigen. We identify conditions that allow phospho-specific biopanning using scFvs with a wide range of affinities (KD = 0.2 to 60 nM). Finally, we demonstrate the capability of screening large libraries by performing biopanning in 6-well plates. These results show that biopanning can effectively select yeast cells based on phospho-site specific antibody binding, opening doors for the facile identification of high-quality monoclonal antibodies.

Depth-multiplexed ptychographic microscopy for high-throughput imaging of stacked bio-specimens on a chip.

Imaging a large number of bio-specimens at high speed is essential for many biomedical applications. The common strategy is to place specimens at different lateral positions and image them sequentially. Here we report a new on-chip imaging strategy, termed depth-multiplexed ptychographic microscopy (DPM), for parallel imaging and sensing at high speed. Different from the common strategy, DPM stacks multiple specimens in the axial direction and images the entire z-stack all at once. In our prototype platform, we modify a low-cost car mirror for programmable steering of the incident laser beam. A blood-coated image sensor is then placed underneath the stacked sample for acquiring the resulting diffraction patterns. With the captured images, we perform blind recovery of the incident beam angle and model different layers of the stacked sample as different coded surfaces for object reconstruction. For in vitro experiment, we demonstrate time-lapse cell culture monitoring by imaging 3 stacked microfluidic channels on the coded sensor. For high-throughput cytometric analysis, we image 5 stacked brain sections with a 205-mm2 field of view in ∼50 s. Cytometric analysis is also performed to quantify the cellular proliferation biomarkers on the slides. The DPM approach adds a new degree of freedom for data multiplexing in microscopy, enabling parallel imaging of multiple specimens using a single detector. The demonstrated 6-mm depth of field is among the longest ones in microscopy imaging. The novel depth-multiplexed configuration also complements the miniaturization provided by microfluidics devices, offering a solution for on-chip sensing and imaging with efficient sample handling.

Degradation-driven protein level oscillation in the yeast Saccharomyces cerevisiae.

Generating robust, predictable perturbations in cellular protein levels will advance our understanding of protein function and enable the control of physiological outcomes in biotechnology applications. Timed periodic changes in protein levels play a critical role in the cell division cycle, cellular stress response, and development. Here we report the generation of robust protein level oscillations by controlling the protein degradation rate in the yeast Saccharomyces cerevisiae. Using a photo-sensitive degron and red fluorescent proteins as reporters, we show that under constitutive transcriptional induction, repeated triangular protein level oscillations as fast as 5-10 min-scale can be generated by modulating the protein degradation rate. Consistent with oscillations generated though transcriptional control, we observed a continuous decrease in the magnitude of oscillations as the input modulation frequency increased, indicating low-pass filtering of input perturbation. By using two red fluorescent proteins with distinct maturation times, we show that the oscillations in protein level is largely unaffected by delays originating from functional protein formation. Our study demonstrates the potential for repeated control of protein levels by controlling the protein degradation rate without altering the transcription rate.

Optogenetics in Sinorhizobium meliloti Enables Spatial Control of Exopolysaccharide Production and Biofilm Structure.

Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.