A randomized library approach to identifying functional lox site domains for the Cre recombinase

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system

Hydrodynamics of membranes: the bilayer aspect and adhesion

We review recent work on the dynamics of membranes. First, we discuss the viscous modes of a free bilayer membrane, taking into account the coupling between bending and the local density of the two monolayers. Apart from important corrections to the conventional bending mode, we obtain a second slow mode which is essentially a fluctuation in the density difference of the two monolayers, damped by intermonolayer friction. Estimates for a stack of membranes show reasonable agreement with a recent spin-echo study of membrane undulations. Second, we discuss the dispersion relation for both fluid and polymerized membranes bound to a substrate by an attractive potential. The interplay between the hydrodynamic damping by the surrounding liquid and the restoring force in the binding potential leads to a q2 dependence of the damping rate γ at small wavevector q. With increasing q, various crossovers can occur, including the possibility of nonmonotonic damping in which γ decreases with q as ∼ 1/q

Viscous modes of fluid bilayer membranes

We determine the dispersion relation for a fluid bilayer membrane, taking into account the coupling between bending and the local density of the two monolayers. Apart from important corrections to the conventional bending mode, we obtain a second slow mode which is essentially a fluctuation in the density difference of the two monolayers, damped by inter-monolayer friction. Estimates for a stack of membranes show reasonable agreement with a recent spin-echo study of membrane undulations

Effective Temperatures of a Driven System Near Jamming

Fluctuations in a model of a sheared, zero-temperature foam are studied numerically. Five different quantities that reduce to the true temperature in an equilibrium thermal system are calculated. All five have the same shear-rate dependence, and three have the same value. Near the onset of jamming, the relaxation time is the same function of these three temperatures in the sheared system as of the true temperature in an unsheared system. These results imply that statistical mechanics is useful for the system and provide strong support for the concept of jamming.Comment: 4 pages, 4 postscript figure

The Melting of Aqueous Foams

Diffusing-wave spectroscopy measurements show that ordinarily solid aqueous foams flow by a series of stick-slip avalanche-like rearrangements of neighboring bubbles from one tight packing configuration to another. Contrary to a recent prediction, the distribution of avalanche sizes do not obey a power-law distribution characteristic of self-organized criticality. This can be understood from a simple model of foam mechanics based on bubble-bubble interactions

Statistics of Shear-Induced Rearrangements in a Two-Dimensional Model Foam

Under steady shear, a foam relaxes stress through intermittent rearrangements of bubbles accompanied by sudden drops in the stored elastic energy. We use a simple model of foam that incorporates both elasticity and dissipation to study the statistics of bubble rearrangements in terms of energy drops, the number of nearest neighbor changes, and the rate of neighbor-switching (T1) events. We do this for a two-dimensional system as a function of system size, shear rate, dissipation mechanism, and gas area fraction. We find that for dry foams, there is a well-defined quasistatic limit at low shear rates where localized rearrangements occur at a constant rate per unit strain, independent of both system size and dissipation mechanism. These results are in good qualitative agreement with experiments on two-dimensional and three-dimensional foams. In contrast, we find for progessively wetter foams that the event size distribution broadens into a power law that is cut off only by system size. This is consistent with criticality at the melting transition

Synthesis and characterisation of rylene diimide dimers using molecular handcuffs

A strategy for positioning, and loosely connecting, molecules in close proximity using mechanically interlocked handcuffs is described. The strategy is demonstrated using rylene diimides, creating dimeric structures in which two components are linked through pillar[5]arene/imidazolium rotaxanes. Investigation of the resulting molecules demonstrates intriguing and new properties that arise from placing these redox active dye molecules together, allowing interactions, whilst allowing the molecules to separate as required. In particular we observe excimer emission from a perylene diimide dimer handcuff and the formation of an unusual radical anion π-dimer upon double reduction of the same molecule. The latter exhibits a unique visible absorption profile for a PDI-based molecule. We demonstrate the flexibility of our approach by making an unprecedented mixed perylene diimide/naphthalene diimide dimer which also reveals interactions between the two components. Our synthetic strategy facilitates the creation of unusual dimeric structures and allows the investigation of intermolecular interactions and the effects they have on electronic and magnetic properties