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.

Draft Genome Assembly of Stutzerimonas sp. Strain S1 and Achromobacter spanius Strain S4, Two Syringol-Metabolizing Bacteria Isolated from Compost Soil.

Two bacterial strains able to use syringol as a sole carbon source were isolated from compost. The isolates, named S1 and S4, were sequenced using the Illumina platform. The final assemblies contained 4.2 Mbp, 63% GC, and 3,912 genes for S1 and 6.2 Mbp, 64% GC, and 5,503 genes for S4.

Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates.

Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient route to produce phenolic amines. Here, a novel cell growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94- and 92-fold increased specific transaminase activity, respectively. Whole-cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA, respectively. Still, amine production was limited by a substantial re-assimilation of the product and formation of the by-products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re-assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.

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 (<10(-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.

Nanoplasmonic probes of catalytic reactions.

Optical probes of heterogeneous catalytic reactions can be valuable tools for optimization and process control because they can operate under realistic conditions, but often probes lack sensitivity. We have developed a plasmonic sensing method for such reactions based on arrays of nanofabricated gold disks, covered by a thin (approximately 10 nanometer) coating (catalyst support) on which the catalyst nanoparticles are deposited. The sensing particles monitor changes in surface coverage of reactants during catalytic reaction through peak shifts in the optical extinction spectrum. Sensitivities to below 10(-3) monolayers are estimated. The capacity of the method is demonstrated for three catalytic reactions, CO and H2 oxidation on Pt, and NO(x) conversion to N2 on Pt/BaO.

A combined nanoplasmonic and electrodeless quartz crystal microbalance setup.

We have developed an instrument combining localized surface plasmon resonance (LSPR) sensing with electrodeless quartz crystal microbalance with dissipation monitoring (QCM-D). The two techniques can be run simultaneously, on the same sensor surface, and with the same time resolution and sensitivity as for the individual techniques. The electrodeless QCM eliminates the need to fabricate electrodes on the quartz crystal and gives a large flexibility in choosing the surface structure and coating for both QCM-D and LSPR. The performance is demonstrated for liquid phase measurements of lipid bilayer formation and biorecognition events, and for gas phase measurements of hydrogen uptake/release by palladium nanoparticles. Advantages of using the combined equipment for biomolecular adsorption studies include synchronized information about structural transformations and extraction of molecular (dry) mass and degree of hydration of the adlayer, which cannot be obtained with the individual techniques. In hydrogen storage studies the combined equipment, allows for synchronized measurements of uptake/release kinetics and quantification of stored hydrogen amounts in nanoparticles and films at practically interesting hydrogen pressures and temperatures.

Sensing characteristics of NIR localized surface plasmon resonances in gold nanorings for application as ultrasensitive biosensors.

The optical responses of 75-150 nm diameter gold nanorings to changes in local refractive index have been quantified by near-infrared extinction spectroscopy and compared to DDA calculations and an analytical approach. The "bulk" refractive index sensitivities of gold nanorings are substantially (>5 times) larger than those of nanodisks with similar diameters. Nanorings retain a significantly larger sensitivity than nanodisks at the same spectral position, demonstrating a clear shape dependence that may correlate to a systematic difference in the influence of the dielectric substrate. The nanoring bulk refractive index sensitivity scales linearly with plasmon peak position. The spectral sensitivity to thin films of alkanethiols gave a shift of approximately 5.2 nm/CH2 unit while bulk sensitivities as high as 880 nm/RIU were observed, the highest such reported sensitivities. Both bulk and thin dielectric film sensitivities correlated well with theory. Real-time label-free monitoring of protein binding via molecular recognition was demonstrated.