Small Science: A Tool and Tips for Converting Food Science Demonstrations into Public Inquiry Experiences

Small-scale science activities provide an opportunity for engagement of diverse, large audiences at settings such as 4-H fairs. We present practical information for implementation of small-scale food science experiences in the Extension education context. Our focus is description of a tool for adaptation of activities for inquiry-based learning and tips for miniaturization of activities to save on costs and resources

Water absorption and degradation characteristics of chitosan-based polyesters and hydroxyapatite composites

Blends of chitosan and biodegradable synthetic aliphatic polyesters (polycaprolactone, poly-(butylene succinate), poly[(butylene succinate)-co-adipate], poly[(butylene terephthalate)-co-adipate], and poly(lactic acid)) were injection-molded. These samples were immersed in isotonic solution at 37ºC for a period of 60 d. The water uptake and the degradation properties, as measured by the loss in tensile strength, were evaluated as a function of time. In this study, the rate and the equilibriumwater uptake were proportional to the amount of chitosan in the blend. The addition of HA to chitosan and polyester significantly reduced the equilibrium water uptake. The water uptake did not follow the classical Fickian phenomena and could be expressed by a two-stage sorption non-Fickian diffusion model. Contact angle measurement was used to quantify the changes in surface hydrophilicity as a function of chitosan and polyester composition. The glycerol contact angle decreased with increasing synthetic components in the blend. The blends and composites also showed increased degradation, as quantified by a loss in their mechanical properties, with increase in natural content. The degradation of properties was directly related to the water uptake of the blends; the higher the water uptake, the higher the degradation. Pure polyesters, while having low water uptake, nevertheless showed significant degradation by a precipitous drop in the strain at break. Among the polyesters, poly(lactic acid) displayed maximum degradation, while polycaprolactone displayed the least.Blends of chitosan and biodegradable synthetic aliphatic polyesters (polycaprolactone, poly(butylene succinate), poly[(butylene succinate)-co-adipate], poly[(butylene terephthalate)co-adipate], and poly(lactic acid)) were injection-molded. These samples were immersed in isotonic solution at 37 degrees C for a period of 60 d. The water uptake and the degradation properties, as measured by the loss in tensile strength, were evaluated as a function of time. In this study, the rate and the equilibrium water uptake were proportional to the amount of chitosan in the blend. The addition of HA to chitosan and polyester significantly reduced the equilibrium water uptake. The water uptake did not follow the classical Fickian phenomena and could be expressed by a two-stage sorption non-Fickian diffusion model. Contact angle measurement was used to quantify the changes in surface hydrophilicity as a function of chitosan and polyester composition. The glycerol contact angle decreased with increasing synthetic components in the blend. The blends and composites also showed increased degradation, as quantified by a loss in their mechanical properties, with increase in natural content. The degradation of properties was directly related to the water uptake of the blends; the higher the water uptake, the higher the degradation. Pure polyesters, while having low water uptake, nevertheless showed significant degradation by a precipitous drop in the strain at break. Among the polyesters, poly(lactic acid) displayed maximum degradation, while polycaprolactone displayed the least

Extensive carbon isotopic heterogeneity among methane seep microbiota

To assess and study the heterogeneity of δ^(13)C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ^(13)C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ^(13)C values (> 50‰) in this microbial community. The δ^(13)C of ANME-1 rods ranged from −24‰ to −87‰. The δ^(13)C of ANME-2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as ^(13)C-depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more ^(13)C-depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ^(13)C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth

Transcription from bacteriophage λ pR promoter is regulated independently and antagonistically by DksA and ppGpp

The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage λ major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in λ phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation

In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme

In vitro reconstitution of a biological complex or process normally involves assembly of multiple individually purified protein components. Here we present a strategy that couples expression and assembly of multiple gene products with functional detection in an in vitro reconstituted protein synthesis system. The strategy potentially allows experimental reconstruction of a multi-component biological complex or process using only DNA templates instead of purified proteins. We applied this strategy to bacterial transcription initiation by co-expressing genes encoding Escherichia coli RNA polymerase subunits and sigma factors in the reconstituted protein synthesis system and by coupling the synthesis and assembly of a functional RNA polymerase holoenzyme with the expression of a reporter gene. Using such a system, we demonstrated sigma-factor-dependent, promoter-specific transcription initiation. Since protein synthesis, complex formation and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles natural biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by conventional reconstitution methods, allow rapid generation and detection of genetic mutations, and provide an open and designable platform for in vitro study and intervention of complex biological processes

Transcription initiation factor DksA has diverse effects on RNA chain elongation

Bacterial transcription factors DksA and GreB belong to a family of coiled-coil proteins that bind within the secondarychannel of RNA polymerase (RNAP). These proteins display structural homology but play different regulatory roles. DksA disrupts RNAP interactions with promoter DNA and inhibits formation of initiation complexes, sensitizing rRNA synthesis to changes in concentrations of ppGpp and NTPs. Gre proteins remodel the RNAP active site and facilitate cleavage of the nascent RNA in elongation complexes. However, DksA and GreB were shown to have overlapping effects during initiation, and in vivo studies suggested that DksA may also function at post-initiation steps. Here we show that DksA has many features of an elongation factor: it inhibits both RNA chain extension and RNA shortening by exonucleolytic cleavage or pyrophosphorolysis and increases intrinsic termination in vitro and in vivo. However, DksA has no effect on Rho- or Mfd-mediated RNA release or nascent RNA cleavage in backtracked complexes, the regulatory target of Gre factors. Our results reveal that DksA effects on elongating RNAP are very different from those of GreB, suggesting that these regulators recognize distinct states of the transcription complex

Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function

Diffusion in Model Networks as Studied by NMR and Fluorescence Correlation Spectroscopy

We have studied the diffusion of small solvent molecules (octane) and larger hydrophobic dye probes in octane-swollen poly(dimethyl siloxane) linear-chain solutions and end-linked model networks, using pulsed-gradient nuclear magnetic resonance (NMR) and fluorescence correlation spectroscopy (FCS), respectively, focusing on diffusion in the bulk polymer up to the equilibrium degree of swelling of the networks, that is, 4.8 at most. The combination of these results allows for new conclusions on the feasibility of different theories describing probe diffusion in concentrated polymer systems. While octane diffusion shows no cross-link dependence, the larger dyes are increasingly restricted by fixed chemical meshes. The simple Fujita free-volume theory proved most feasible to describe probe diffusion in linear long-chain solutions with realistic parameters, while better fits were obtained assuming a stretched exponential dependence on concentration. Importantly, we have analyzed the cross-link specific effect on probe diffusion independently of any specific model by comparing the best-fit interpolation of the solution data with the diffusion in the networks. The most reasonable description is obtained by assuming that the cross-link effect is additive in the effective friction coefficient of the probes. The concentration dependences as well as the data compared at the equilibrium degrees of swelling indicate that swelling heterogeneities and diffusant shape have a substantial influence on small-molecule diffusion in networks.