E. Coli and oxygen: a motility transition.

The motility of Escherichia coli is correlated with oxygen concentration. We show that oxygen penetrating into an anaerobic sample induces the coexistence of two domains of motile and nonmotile bacteria. This coexistence generates a bacterial accumulation at the border that propagates slowly with a constant velocity. To show that this front propagation follows general scaling arguments, we characterize the sharp and fast motile to nonmotile transition occurring when bacteria exhaust oxygen. Additionally, we develop a novel technique to quantify oxygen in situ without affecting bacteria.

Solitary modes of bacterial culture in a temperature gradient.

We study the behavior of a bacterial culture in a one-dimensional temperature gradient. The bacteria first accumulate near their natural temperature due to thermotaxis. The maximum of the bacterial density profile then drifts to lower temperature with a velocity proportional to the initial concentration of bacteria (typical velocity 0.5 microm/sec). Above a critical concentration of 10(8) cells/cm(3), a new mode develops from the initial accumulation in the form of a sharp pulse moving at a faster velocity ( approximately 3.5 microm/sec). The time of development of this mode diverges as the concentration approaches its critical value. This mode is a result of a positive feedback mechanism provided by interbacterial communication. A theoretical model shows good agreement with the experimental results.

Effects of DNA sequence and structure on binding of RecA to single-stranded DNA.

Fluorescence anisotropy is used to follow the binding of RecA to short single-stranded DNA (ssDNA) sequences (39 bases) at low DNA and RecA concentration where the initial phase of polymerization occurs. We observe that RecA condensation is extremely sensitive to minute changes in DNA sequences. RecA binds strongly to sequences that are rich in pyrimidines and that lack significant secondary structure and base stacking. We find a correlation between the DNA folding free energy and the onset concentration for RecA binding. These results suggest that the folding of ssDNA and base stacking represent a barrier for RecA binding. The link between secondary structure and binding affinity is further analyzed with two examples: discrimination between two naturally occurring polymorphisms differing by one base and RecA binding on a molecular beacon. A self-assembly model is introduced to explain these observations. We propose that RecA may be used to sense ssDNA sequence and structure.

Dynamics of DNA-protein interaction deduced from in vitro DNA evolution.

We report the first experimental study of the dynamics of in vitro evolution of DNA. Starting from a random pool of DNA sequences, we used cycles of selection (binding to the lac repressor protein), amplification, and mutations to evolve to a unique DNA sequence, the lac operator. Statistical analysis of the DNA sequences obtained during the cycles of evolution shows that the DNA bases are selected at different rates. The rates of selection provide a quantitative measure of the interaction of the DNA bases with the protein during the complex formation. A model reproduces the evolution dynamics of the DNA population but cannot give the fine structure of the DNA-protein interaction.

Single-mismatch detection using gold-quenched fluorescent oligonucleotides.

Here we describe a hybrid material composed of a single-stranded DNA (ssDNA) molecule, a 1.4 nm diameter gold nanoparticle, and a fluorophore that is highly quenched by the nanoparticle through a distance-dependent process. The fluorescence of this hybrid molecule increases by a factor of as much as several thousand as it binds to a complementary ssDNA. We show that this composite molecule is a different type of molecular beacon with a sensitivity enhanced up to 100-fold. In competitive hybridization assays, the ability to detect single mismatch is eightfold greater with this probe than with other molecular beacons.

Flexible filaments in a flowing soap film as a model for one-dimensional flags in a two-dimensional wind.

The dynamics of swimming fish and flapping flags involves a complicated interaction of their deformable shapes with the surrounding fluid flow. Even in the passive case of a flag, the flag exerts forces on the fluid through its own inertia and elastic responses, and is likewise acted on by hydrodynamic pressure and drag. But such couplings are not well understood. Here we study these interactions experimentally, using an analogous system of flexible filaments in flowing soap films. We find that, for a single filament (or 'flag') held at its upstream end and otherwise unconstrained, there are two distinct, stable dynamical states. The first is a stretched-straight state: the filament is immobile and aligned in the flow direction. The existence of this state seems to refute the common belief that a flag is always unstable and will flap. The second is a flapping state: the filament executes a sinuous motion in a manner akin to the flapping of a flag in the wind. We study further the hydrodynamically coupled interaction between two such filaments, and demonstrate the existence of four different dynamical states.

Method for linking a synthesized protein to its mRNA-DNA complex.

A nascent protein remains in a complex with its ribosome and mRNA if the stop codon is deleted from the mRNA. In the same manner, mRNA forms a stable complex with DNA if the transcription termination is blocked. In principle, if both mRNA translation and DNA transcription termination are prevented, the protein should stay in a complex with its mRNA and DNA. A method is designed to test these possibilities. Using an immobilized luciferase gene sequence, a functional luciferase protein is produced that remains associated through its mRNA with its DNA, confirming the feasibility of the proposed scheme. It has potential application for in vitro synthesis of proteins and protein micro-arrays.

Particle diffusion in a quasi-two-dimensional bacterial bath.

We study the effect of bacterial motion on micron-scale beads in a freely suspended soap film. Given the sizes of bacteria and beads, the geometry of the experiment is quasi-two-dimensional. Large positional fluctuations are observed for beads as large as 10 microm in diameter, and the measured mean-square displacements indicate superdiffusion in short times and normal diffusion in long times. Though the phenomenon is similar to Brownian motions of small particles, its physical origin is different and can be attributed to the collective dynamics of bacteria.

Periodic boundary motion in thermal turbulence

A free-floating plate is introduced in a Benard convection cell with an open surface. It partially covers the cell and distorts the local heat flux, inducing a coherent flow that in turn moves the plate. Remarkably, the plate can be driven to a periodic motion even under the action of a turbulent fluid. The period of the oscillation depends on the coverage ratio, and on the Rayleigh number of the convective system. The plate oscillatory behavior observed in this experiment may be related to a geological model, in which continents drift in a quasiperiodic fashion.

Sequence dependent rigidity of single stranded DNA.

Single stranded DNA (ssDNA) equilibrium dynamics are investigated using a fluorophore/quencher-labeled hairpin structure which thermally fluctuates between open and closed states. Temporal correlations of the fluorescence fluctuations are used to determine the energy barrier to conformational change. We find that ssDNA distortion is purely entropic for poly(T) but requires an additional enthalpy of +0.5 kcal x mol(-1) x base(-1) for poly(A), consistent with the disruption of base stacking. Such sequence dependent dynamics challenge the classical model of ssDNA as a completely flexible coil.

RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis.

The polymerization of RecA on individual double-stranded DNA molecules is studied. A linear DNA (lambda DNA, 48.5 Kb), anchored at one end to a cover glass and at the other end to an optically trapped 3-micrometers diameter polystyrene bead, serves as a template. The elongation caused by RecA assembly is measured in the presence of ATP and ATP[gammaS]. By using force extension and hydrodynamic recoil, a value of the persistence length of the RecA-DNA complex is obtained. In the presence of ATP, the polymer length is unstable, first growing to saturation and then decreasing. This suggests a transient dynamics of association and dissociation for RecA on a double-stranded DNA, the process being controlled by ATP hydrolysis. Part of this dynamics is suppressed in the presence of ATP[gammaS], leading to a stabilized RecA-DNA complex. A one-dimensional nucleation and growth model is presented that may account for the protein assembly.

Thermodynamic basis of the enhanced specificity of structured DNA probes.

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

Kinetics of conformational fluctuations in DNA hairpin-loops.

The kinetics of DNA hairpin-loop fluctuations has been investigated by using a combination of fluorescence energy transfer and fluorescence correlation spectroscopy. We measure the chemical rates and the activation energies associated with the opening and the closing of the hairpin for different sizes and sequences of the loop and for various salt concentrations. The rate of unzipping of the hairpin stem is essentially independent of the characteristics of the loop, whereas the rate of closing varies greatly with the loop length and sequence. The closing rate scales with the loop length, with an exponent 2.6 +/- 0.3. The closing rate is increased at higher salt concentrations. For hairpin closing, a loop of adenosine repeats leads to smaller rates and higher activation energies than a loop with thymine repeats.

Gramicidin channel kinetics under tension.

We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

Elasticity and structure of eukaryote chromosomes studied by micromanipulation and micropipette aspiration.

The structure of mitotic chromosomes in cultured newt lung cells was investigated by a quantitative study of their deformability, using micropipettes. Metaphase chromosomes are highly extensible objects that return to their native shape after being stretched up to 10 times their normal length. Larger deformations of 10 to 100 times irreversibly and progressively transform the chromosomes into a "thin filament," parts of which display a helical organization. Chromosomes break for elongations of the order of 100 times, at which time the applied force is around 100 nanonewtons. We have also observed that as mitosis proceeds from nuclear envelope breakdown to metaphase, the native chromosomes progressively become more flexible. (The elastic Young modulus drops from 5,000 +/- 1,000 to 1,000 +/- 200 Pa.) These observations and measurements are in agreement with a helix-hierarchy model of chromosome structure. Knowing the Young modulus allows us to estimate that the force exerted by the spindle on a newt chromosome at anaphase is roughly one nanonewton.

Parallel overlap assembly for the construction of computational DNA libraries.

Algorithms for computing with DNA currently require the construction of pools of molecules in which each distinct molecule represents a different starting point for the calculation. We have begun building such pools using the technique of parallel overlap assembly that is already used for the generation of diversity in biologically useful combinatorial search techniques such as gene shuffling. Unlike these applications, a pool in a molecular computer must be complete, containing all possible strands, and ordered, having minimal contamination from incorrectly assembled DNA. We present an experiment in which parallel overlap assembly is used to construct a computational pool and an experiment in which this pool is used to solve the NP-complete maximal-clique problem.

DNA solution of the maximal clique problem.

The maximal clique problem has been solved by means of molecular biology techniques. A pool of DNA molecules corresponding to the total ensemble of six-vertex cliques was built, followed by a series of selection processes. The algorithm is highly parallel and has satisfactory fidelity. This work represents further evidence for the ability of DNA computing to solve NP-complete search problems.