The effects of the environment on the stability and expression of genetically engineered plasmids

The lac promoter is widely used in plasmid expression systems, even though it is prone to catabolite repression. As a consequence glycerol is often used as an alternative carbon source. Three plasmids containing various sizes of the staphylococcal protein A (SPA) gene, which are under the control of the lac promoter were investigated in continuous culture, to evaluate the effects of nutrient limitations on their stability and expression. The fears of catabolite repression were dispelled as a low expression plasmid (pPA16) produced a greater amount of truncated SPA under glucose limiting conditions (11 ug mg-1 cell protein) when compared to that using glycerol (8 ug mg-1 cell protein). Segregational instability was also observed under glycerol limiting conditions at all the dilution rates investigated. Whereas pPA16 was relatively stable under glucose limiting conditions, with SPA production being continuous. Experiments using excess glycerol with limited ammonium increased the stability of pPA16, (when compared to limited glycerol) with expression of SPA being continuous but reduced (6 ug mg-1 cell protein). With excess glucose and limited ammonium the copy numbers remained high but expression of SPA paralled that produced under glucose limiting conditions. This might indicate that the higher levels of glucose are reducing expression (catabolite repression) or that the low level of ammonium is affecting protein production. A high expression plasmid (pPA31) produced nearly 100 ug full length SPA mg-1 cell protein, while another high expression plasmid (pPA34) producing truncated SPA proved to be very unstable. An ELISA was developed to detect the SPA produced by these experiments, which could be adapted for western blotting or immunogold probing using electron microscopy. SPA was localised in electron lucent areas present in the periplasmic space of the E. coli host harbouring pPA16. While in the same host containing pPA31, SPA was localised not only in electron lucent areas but also around the whole of the outer-membrane.

Hydrotalcite intercalated siRNA:computational characterization of the interlayer environment

Using molecular dynamics (MD) simulations, we explore the structural and dynamical properties of siRNA within the intercalated environment of a Mg:Al 2:1 Layered Double Hydroxide (LDH) nanoparticle. An ab initio force field (Condensed-phase Optimized Molecular Potentials for Atomistic Simulation Studies: COMPASS) is used for the MD simulations of the hybrid organic-inorganic systems. The structure, arrangement, mobility, close contacts and hydrogen bonds associated with the intercalated RNA are examined and contrasted with those of the isolated RNA. Computed powder X-ray diffraction patterns are also compared with related LDH-DNA experiments. As a method of probing whether the intercalated environment approximates the crystalline or rather the aqueous state, we explore the stability of the principle parameters (e.g., the major groove width) that differentiate both A- and A'- crystalline forms of siRNA and contrast this with recent findings for the same siRNA simulated in water. We find the crystalline forms remain structurally distinct when intercalated, whereas this is not the case in water. Implications for the stability of hybrid LDH-RNA systems are discussed.

The performance of stabilized and unstabilized minestone in an aqueous environment with emphasis on its erosion resistance

Britain's sea and flood defences are becoming increasingly aged and as a consequence, more fragile and vulnerable. As the government's philosophy on resources shifts against the use of prime quarried and dredged geo-materials, the need to find alternative bulk materials to bolster Britain's prone defences becomes more pressing. One conceivable source for such a material is colliery waste or minestone. Although a plethora of erosion-abrasion studies have been carried out on soils and soil-cements, very little research has been undertaken to determine the resistance of minestone and its cement stabilized form to the effects of water erosion. The thesis reviews the current extent to which soil-cements, minestone and cement stabilized minestone (CSM) have been employed for hydraulic construction projects. A synopsis is also given on the effects of immersion on shales, mudstones and minestone, especially with regard to the phenomena of slaking. A laboratory study was undertaken featuring a selection of minestones from several British coalfields. The stability of minestone and CSM in sea water and distilled water was assessed using slaking tests and immersion monitoring and the bearing on the use of these materials for hydraulic construction is discussed. Following a review of current erosion apparatus, the erosion/abrasion test and rotating cylinder device were chosen and employed to assess the erosion resistance of minestone and CSM. Comparison was made with a sand mix designed to represent a dredged sand, the more traditional, bulk hydraulic construction material. The results of the erosion study suggest that both minestone and CSM were more resistant to erosion and abrasion than equivalently treated sand mixes. The greater resistance of minestone to the agents of erosion and abrasion is attributed to several factors including the size of the particles, a greater degree of cement bonding and the ability of the minestone aggregate to absorb, rather than transmit shock waves produced by impacting abrasive particles. Although minestone is shown to be highly unstable when subjected to cyclic changes in its moisture content, the study suggests that even in an intertidal regime where cyclic immersion does takes place, minestone will retain sufficient moisture within its fabric to prevent slaking from taking place. The slaking study reveals a close relationship between slaking susceptibility and total pore surface area as revealed by porosimetry. The immersion study shows that although the fabric of CSM is rapidly attacked in sea water, a high degree of the disruption is associated with the edges and corners of samples (ie. free surface) while the integrity of the internal fabric remains relatively intact. CSM samples were shown to be resilient when subjected to immersion in distilled water. An overall assessment of minestone and CSM would suggest that with the implementation of judicious selection and appropriate quality control they could be used as alternative materials for flood and sea defences. It is believed, that even in the harsh regime of a marine environment, CSM could be employed for temporary and sacrificial schemes.

An investigation into the wavelength stability of polymer optical fibre Bragg gratings

The inscription of Bragg gratings has been demonstrated in PMMA-based polymer optical fibre. The water affinity of PMMA can introduce significant wavelength change in a polymer optical fibre Bragg grating (POFBG). In polymer optical fibre losses are much higher than with silica fibre. Very strong absorption bands related to higher harmonics of vibrations of the C-H bond dominate throughout the visible and near infrared. Molecular vibration in substances generates heat, which is referred to as the thermal effect of molecular vibration. This means that a large part of the absorption of optical energy in those spectral bands will convert into thermal energy, which eventually drives water content out of the polymer fibre and reduces the wavelength of POFBG. In this work we have investigated the wavelength stability of POFBGs in different circumstances. The experiment has shown that the characteristic wavelength of a POFBG starts decreasing after a light source is applied to it. This decrease continues until equilibrium inside the fibre is established, depending on the initial water content inside the fibre, the surrounding humidity, the optical power applied, and the fibre size. Our investigation has shown that POFBGs operating at around 850 nm show much smaller wavelength reduction than those operating at around 1550 nm in the same fibre; POFBGs with different diameters show different changes; POFBGs powered by a low level light source, or operating in a very dry environment are least affected by this thermal effect.

An investigation into the wavelength stability of polymer optical fibre Bragg gratings

The inscription of Bragg gratings has been demonstrated in PMMA-based polymer optical fibre. The water affinity of PMMA can introduce significant wavelength change in a polymer optical fibre Bragg grating (POFBG). In polymer optical fibre losses are much higher than with silica fibre. Very strong absorption bands related to higher harmonics of vibrations of the C-H bond dominate throughout the visible and near infrared. Molecular vibration in substances generates heat, which is referred to as the thermal effect of molecular vibration. This means that a large part of the absorption of optical energy in those spectral bands will convert into thermal energy, which eventually drives water content out of the polymer fibre and reduces the wavelength of POFBG. In this work we have investigated the wavelength stability of POFBGs in different circumstances. The experiment has shown that the characteristic wavelength of a POFBG starts decreasing after a light source is applied to it. This decrease continues until equilibrium inside the fibre is established, depending on the initial water content inside the fibre, the surrounding humidity, the optical power applied, and the fibre size. Our investigation has shown that POFBGs operating at around 850 nm show much smaller wavelength reduction than those operating at around 1550 nm in the same fibre; POFBGs with different diameters show different changes; POFBGs powered by a low level light source, or operating in a very dry environment are least affected by this thermal effect.