Development of Cell-Free Transcription–Translation Systems in Three Soil Pseudomonads

In vitro transcription–translation (TX–TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX–TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens. All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX–TL systems, P. synxantha, demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. A set of different constitutive promoters driving mNeonGreen expression were tested in TX–TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX–TL-derived promoter strength and the in vivo promoter strength indicates that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design–build–test cycle

Development of Cell-Free Transcription–Translation Systems in Three Soil Pseudomonads

In vitro transcription–translation (TX–TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX–TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens. All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX–TL systems, P. synxantha, demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. A set of different constitutive promoters driving mNeonGreen expression were tested in TX–TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX–TL-derived promoter strength and the in vivo promoter strength indicates that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design–build–test cycle

Development of Cell-Free Transcription–Translation Systems in Three Soil Pseudomonads

In vitro transcription–translation (TX–TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX–TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens. All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX–TL systems, P. synxantha, demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. A set of different constitutive promoters driving mNeonGreen expression were tested in TX–TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX–TL-derived promoter strength and the in vivo promoter strength indicates that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design–build–test cycle

Engineering the Soil Bacterium Pseudomonas synxantha 2–79 into a Ratiometric Bioreporter for Phosphorus Limitation

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty in ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2–79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25–100 μM without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8–8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporter’s performance in shaken liquid culture predicts its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally aware design process provides an example of how laboratory bioengineering efforts can generate microbes with a greater promise to function reliably in their applied contexts

Indirect Nanoplasmonic Sensing: Ultrasensitive Experimental Platform for Nanomaterials Science and Optical Nanocalorimetry

Indirect nanoplasmonic sensing is a novel experimental platform for measurements of thermodynamics and kinetics in/on nanomaterials and thin films. It features simple experimental setup, high sensitivity, small sample amounts, high temporal resolution (−3 s), operating conditions from UHV to high pressure, wide temperature range, and applicability to any nano- or thin film material. The method utilizes two-dimensional arrangements of nanoplasmonic Au sensor-nanoparticles coated with a thin dielectric spacer layer onto which the sample material is deposited. The measured signal is spectral shifts of the Au-sensor localized plasmons, induced by processes in/on the sample material. Here, the method is applied to three systems exhibiting nanosize effects, (i) the glass transition of confined polymers, (ii) catalytic light-off on Pd nanocatalysts, and (iii) thermodynamics and kinetics of hydrogen uptake/release in Pd nanoparticles <5 nm. In (i) and (iii), dielectric changes in the sample are detected, while (ii) demonstrates a novel optical nanocalorimetry method

Real Time Indirect Nanoplasmonic in Situ Spectroscopy of Catalyst Nanoparticle Sintering

Catalyst deactivation by sintering significantly reduces productivity and energy efficiency of the chemical industry and the effectiveness of environmental cleanup processes. It also hampers the introduction of novel energy conversion devices such as fuel cells. The use of experimental techniques that allow the scrutiny of sintering in situ at high temperatures and pressures in reactive environments is a key to alleviate this situation. Today, such techniques are, however, lacking. Here, we demonstrate by monitoring the sintering kinetics of a Pt/SiO2 model catalyst under such conditions in real time that indirect nanoplasmonic sensing (INPS) has the potential to fill this gap. Specifically, we show an unambiguous correlation between the optical response of the INPS sensor and catalyst sintering. The obtained data are analyzed by means of a kinetic model accounting for the particle-size-dependent activation energy of the Pt detachment. Ostwald ripening is identified as the main sintering mechanism

Real Time Indirect Nanoplasmonic in Situ Spectroscopy of Catalyst Nanoparticle Sintering

Real Time Indirect Nanoplasmonic in Situ Spectroscopy of Catalyst Nanoparticle Sinterin

Environmental Transmission Electron Microscopy Study of the Origins of Anomalous Particle Size Distributions in Supported Metal Catalysts

In this Environmental Transmission Electron Microscopy (ETEM) study we examined the growth patterns of uniform distributions of nanoparticles (NPs) using model catalysts. Pt/SiO2 was heated at 550 °C in 560 Pa of O2 while Pd/carbon was heated in vacuum at 500 °C and in 300 Pa of 5%H2 in Argon at temperatures up to 600 °C. Individual NPs of Pd were tracked to determine the operative sintering mechanisms. We found anomalous growth of NPs occurred during the early stages of catalyst sintering wherein some particles started to grow significantly larger than the mean, resulting in a broadening of the particle size distribution (PSD). The abundance of the larger particles did not fit the log-normal distribution. We can rule out sample nonuniformity as a cause for the growth of these large particles, since images were recorded prior to heat treatments. The anomalous growth of these particles may help explain PSDs in heterogeneous catalysts which often show particles that are significantly larger than the mean, resulting in a long tail to the right. It has been suggested previously that particle migration and coalescence could be the likely cause for such broad size distributions. We did not detect any random migration of the NPs leading to coalescence. A directed migration process was seen to occur at elevated temperatures for Pd/carbon under H2. This study shows that anomalous growth of NPs can occur under conditions where Ostwald ripening is the primary sintering mechanism

Environmental Transmission Electron Microscopy Study of the Origins of Anomalous Particle Size Distributions in Supported Metal Catalysts

In this Environmental Transmission Electron Microscopy (ETEM) study we examined the growth patterns of uniform distributions of nanoparticles (NPs) using model catalysts. Pt/SiO₂ was heated at 550 °C in 560 Pa of O₂ while Pd/carbon was heated in vacuum at 500 °C and in 300 Pa of 5%H₂ in Argon at temperatures up to 600 °C. Individual NPs of Pd were tracked to determine the operative sintering mechanisms. We found anomalous growth of NPs occurred during the early stages of catalyst sintering wherein some particles started to grow significantly larger than the mean, resulting in a broadening of the particle size distribution (PSD). The abundance of the larger particles did not fit the log-normal distribution. We can rule out sample nonuniformity as a cause for the growth of these large particles, since images were recorded prior to heat treatments. The anomalous growth of these particles may help explain PSDs in heterogeneous catalysts which often show particles that are significantly larger than the mean, resulting in a long tail to the right. It has been suggested previously that particle migration and coalescence could be the likely cause for such broad size distributions. We did not detect any random migration of the NPs leading to coalescence. A directed migration process was seen to occur at elevated temperatures for Pd/carbon under H₂. This study shows that anomalous growth of NPs can occur under conditions where Ostwald ripening is the primary sintering mechanism

Environmental Transmission Electron Microscopy Study of the Origins of Anomalous Particle Size Distributions in Supported Metal Catalysts

In this Environmental Transmission Electron Microscopy (ETEM) study we examined the growth patterns of uniform distributions of nanoparticles (NPs) using model catalysts. Pt/SiO₂ was heated at 550 °C in 560 Pa of O₂ while Pd/carbon was heated in vacuum at 500 °C and in 300 Pa of 5%H₂ in Argon at temperatures up to 600 °C. Individual NPs of Pd were tracked to determine the operative sintering mechanisms. We found anomalous growth of NPs occurred during the early stages of catalyst sintering wherein some particles started to grow significantly larger than the mean, resulting in a broadening of the particle size distribution (PSD). The abundance of the larger particles did not fit the log-normal distribution. We can rule out sample nonuniformity as a cause for the growth of these large particles, since images were recorded prior to heat treatments. The anomalous growth of these particles may help explain PSDs in heterogeneous catalysts which often show particles that are significantly larger than the mean, resulting in a long tail to the right. It has been suggested previously that particle migration and coalescence could be the likely cause for such broad size distributions. We did not detect any random migration of the NPs leading to coalescence. A directed migration process was seen to occur at elevated temperatures for Pd/carbon under H₂. This study shows that anomalous growth of NPs can occur under conditions where Ostwald ripening is the primary sintering mechanism