High-throughput optofluidic screening for improved microbial cell factories via real-time micron-scale productivity monitoring.

The industrial synthetic biology sector has made huge investments to achieve relevant miniaturized screening systems for scalable fermentation. Here we present the first example of a high-throughput (>103 genotypes per week) perfusion-based screening system to improve small-molecule secretion from microbial strains. Using the Berkeley Lights Beacon® system, the productivity of each strain could be directly monitored in real time during continuous culture, yielding phenotypes that correlated strongly (r2 > 0.8, p < 0.0005) with behavior in industrially relevant bioreactor processes. This method allows a much closer approximation of a typical fed-batch fermentation than conventional batch-like droplet or microplate culture models, in addition to rich time-dependent data on growth and productivity. We demonstrate these advantages by application to the improvement of high-productivity strains using whole-genome random mutagenesis, yielding mutants with substantially improved (by up to 85%) peak specific productivities in bioreactors. Each screen of ∼5 × 103 mutants could be completed in under 8 days (including 5 days involving user intervention), saving ∼50-75% of the time required for conventional microplate-based screening methods.

Single cell transfection with single molecule resolution using a synthetic nanopore.

We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient but also transfects with single molecule resolution at low voltage (1 V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precision--no other method offers such capabilities.

Biological noise abatement: coordinating the responses of autonomous bacteria in a synthetic biofilm to a fluctuating environment using a stochastic bistable switch.

Noise is inherent to single cell behavior. Its origins can be traced to the stochasticity associated with a few copies of genes and low concentrations of protein and ligands. We have studied the mechanisms by which the response of noisy elements can be entrained for biological signal processing. To elicit predictable biological function, we have engineered a gene environment that incorporates a gene regulatory network with the stringently controlled microenvironment found in a synthetic biofilm. The regulatory network leverages the positive feedback found in quorum-sensing regulatory components of the lux operon, which is used to coordinate cellular responses to environmental fluctuations. Accumulation of the Lux receptor in cells, resulting from autoregulation, confers a rapid response and enhanced sensitivity to the quorum-sensing molecule that is retained after cell division as epigenetic memory. The memory of the system channels stochastic noise into a coordinated response among quorum-sensing signal receivers in a synthetic biofilm in which the noise diminishes with repeated exposure to noisy transmitters on the input of a signaling cascade integrated into the same biofilm. Thus, gene expression in the receivers, which are autonomous and do not communicate with each other, is synchronized to fluctuations in the environment.

Epigenetic memory emerging from integrated transcription bursts.

Some autonomous bacteria coordinate their actions using quorum-sensing (QS) signals to affect gene expression. However, noise in the gene environment can compromise the cellular response. By exercising precise control over a cell's genes and its microenvironment, we have studied the key positive autoregulation element by which the lux QS system integrates noisy signals into an epigenetic memory. We observed transcriptional bursting of the lux receptor in cells stimulated by near-threshold levels of QS ligand. The bursts are integrated over time into an epigenetic memory that confers enhanced sensitivity to the ligand. An emergent property of the system is manifested in pattern formation among phenotypes within a chemical gradient.

Direct visualization of single-molecule translocations through synthetic nanopores comparable in size to a molecule.

A nanopore is the ultimate analytical tool. It can be used to detect DNA, RNA, oligonucleotides, and proteins with submolecular sensitivity. This extreme sensitivity is derived from the electric signal associated with the occlusion that develops during the translocation of the analyte across a membrane through a pore immersed in electrolyte. A larger occluded volume results in an improvement in the signal-to-noise ratio, and so the pore geometry should be made comparable to the size of the target molecule. However, the pore geometry also affects the electric field, the charge density, the electro-osmotic flow, the capture volume, and the response time. Seeking an optimal pore geometry, we tracked the molecular motion in three dimensions with high resolution, visualizing with confocal microscopy the fluorescence associated with DNA translocating through nanopores with diameters comparable to the double helix, while simultaneously measuring the pore current. Measurements reveal single molecules translocating across the membrane through the pore commensurate with the observation of a current blockade. To explain the motion of the molecule near the pore, finite-element simulations were employed that account for diffusion, electrophoresis, and the electro-osmotic flow. According to this analysis, detection using a nanopore comparable in diameter to the double helix represents a compromise between sensitivity, capture volume, the minimum detectable concentration, and response time.

Using a nanopore for single molecule detection and single cell transfection.

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

Sample cells for probing solid/liquid interfaces with broadband sum-frequency-generation spectroscopy.

Two sample cells designed specifically for sum-frequency-generation (SFG) measurements at the solid/liquid interface were developed: one thin-layer analysis cell allowing measurement of films on reflective metallic surfaces through a micrometer layer of solution and one spectroelectrochemical cell allowing investigation of processes at the indium tin oxide/solution interface. Both sample cells are described in detail and data illustrating the capabilities of each are shown. To further improve measurements at solid/liquid interfaces, the broadband SFG system was modified to include a reference beam which can be measured simultaneously with the sample signal, permitting background correction of SFG spectra in real time. Sensitivity tests of this system yielded a signal-to-noise ratio of 100 at a surface coverage of 0.2 molecules/nm(2). Details on data analysis routines, pulse shaping methods of the visible beam, as well as the design of a purging chamber and sample stage setup are presented. These descriptions will be useful to those planning to set up a SFG spectrometer or seeking to optimize their own SFG systems for measurements of solid/liquid interfaces.

In situ characterization of thermo-responsive poly(N-isopropylacrylamide) films with sum-frequency generation spectroscopy.

The thermo-responsive behaviour of thiol modified poly(N-isopropylacrylamide) (pNIPAM) films immobilized on gold are probed by in situ broadband sum-frequency generation (SFG) spectroscopy. The pNIPAM films were prepared by atom transfer radical polymerization (ATRP) using a nitro-biphenyl-thiol (NBT)-SAM on a polycrystalline gold surface as a substrate. Additionally, Raman and infrared reflection absorption spectroscopy (IRRAS) are applied to spin-coated pNIPAM films. Molecular groups involved in the reorientation and disordering of the polymer chains during the LCST (lower critical solution temperature) transition of pNIPAM are identified. The characteristic vibrations of the CH(3) groups show a gradual reorientation of the isopropyl groups within the pNIPAM film and instantaneous reorientation of the outermost CH(3) groups around 32 degrees C.

Sum-frequency-generation spectroscopy of DNA films in air and aqueous environments.

Understanding the organization and orientation of surface-immobilized single stranded DNA (ssDNA) in aqueous environments is essential for optimizing and further developing the technology based on oligonucleotide binding. Here the authors demonstrate how sum-frequency-generation (SFG) spectroscopy can be used to compare the structure and orientation of model monolayers of ssDNA on gold in air, D(2)O, and phosphate buffered saline (PBS) solution. Films of adenine and thymine homo-oligonucleotides showed significant conformational changes in air versus aqueous environments in the CH stretching region. The thymine films showed changes between D(2)O and PBS solution, whereas the SFG spectra of adenine films under these conditions were largely similar, suggesting that the thymine films undergo greater conformational changes than the adenine films. Examination of thymine films in the amide I vibrational region revealed that molecules in films of nonthiolated DNA were lying down on the gold surface whereas molecules in films of thiol-linked DNA were arranged in a brushlike structure. Comparison of SFG spectra in the amide I region for thiol-linked DNA films in air and D(2)O also revealed substantial conformational changes.