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Albert Libchaber

Publications and source records attributed to Albert Libchaber.

17 recordsLinked to original sources
PubMed2006

Examining how the spatial organization of chromatin signals influences metaphase spindle assembly.

During cell division, the proper assembly of a microtubule-based bipolar spindle depends on signals from chromatin. However, it is unknown how the spatial organization of chromatin signals affects spindle morphology. Here, we use paramagnetic chromatin beads, and magnetic fields for their alignment in cell-free extracts, to examine the spatial components of signals that regulate spindle assembly. We find that for linear chromatin-bead arrays that vary by eightfold in length, metaphase spindle size and shape are constant. Our findings indicate that, although chromatin provides cues for microtubule formation, metaphase spindle organization, which is controlled by microtubule-based motors, is robust to changes in the shape of chromatin signals.

Animals↗
PubMed2006

Some aspects of E. coli promoter evolution observed in a molecular evolution experiment.

We devised a molecular evolution procedure to evolve E. coli promoter sequences and applied it to observe an arbitrary, nonfunctional sequence evolving into functional promoters. In the experiments, DNA sequence variations were generated with error-prone PCR and were inserted in the promoter region of the cat (chloramphenicol acetyl transferase) gene on a plasmid. Upon transforming the cells, functional promoters on the plasmid were selected according to the chloramphenicol resistance. Within a few cycles of mutation-selection, promoters emerged, and the sequences converged into a small number of groups. In the process, the extended minus 10 type of promoters emerged quickly, and small deletions were often involved in adjusting the length between the -35 and the -10 elements. Our results also suggest a possible selection for promoter stability against mutation.

Base Sequence↗
PubMed2005

Toward an artificial cell based on gene expression in vesicles.

We present a new experimental approach to build an artificial cell using the translation machinery of a cell-free expression system as the hardware and a DNA synthetic genome as the software. This approach, inspired by the self-replicating automata of von Neumann, uses cytoplasmic extracts, encapsulated in phospholipid vesicles, to assemble custom-made genetic circuits to develop the functions of a minimal cell. Although this approach can find applications, especially in biotechnology, the primary goal is to understand how a DNA algorithm can be designed to build an operating system that has some of the properties of life. We provide insights on this cell-free approach as well as new results to transform step by step a long-lived vesicle bioreactor into an artificial cell. We show how the green fluorescent protein can be anchored to the membrane and we give indications of a possible insertion mechanism of integral membrane proteins. With vesicles composed of different phospholipids, the fusion protein alpha-hemolysin-eGFP can be expressed to reveal patterns on the membrane. The specific degradation complex ClpXP from E. coli is introduced to create a sink for the synthesized proteins. Perspectives and subsequent limitations of this approach are discussed.

Cell-Free System↗
PubMed2004

High-fidelity DNA sensing by protein binding fluctuations.

One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response. We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of-equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.

Adenosine Triphosphate↗
PubMed2004

A vesicle bioreactor as a step toward an artificial cell assembly.

An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles containing extracts are produced in an oil-extract emulsion. To form a bilayer the vesicles are transferred into a feeding solution that contains ribonucleotides and amino acids. Transcription-translation of plasmid genes is isolated in the vesicles. Whereas in bulk solution expression of enhanced GFP stops after 2 h, inside the vesicle permeability of the membrane to the feeding solution prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the alpha-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein production of 30 muM after 4 days. Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst.

Amino Acids↗
PubMed2004

Single-molecule measurements of gold-quenched quantum dots.

We report the study of the quenching of quantum dots (CdSe) by gold nanoparticles at the single-molecule level. Double-stranded DNA is used as a rigid spacer to tune the distance between the two nanoparticles. The width of the fluorescent intensity distribution, monitored at different interparticle distances, reflects both the nanoparticle heterogeneity and the fluorescence intermittency of the quantum dot. The fluorescence distribution emitted by single CdSe nanocrystals can easily be distinguished from the fluorescence of partially quenched CdSe. Our results show that the distance-dependence quenching is compatible with a Förster-type process.

Cadmium Compounds↗
PubMed2004

Discerning aggregation in homogeneous ensembles: a general description of photon counting spectroscopy in diffusing systems.

In order to discern aggregation in solution, we present a quantum mechanical analog of the photon statistics from fluorescent molecules diffusing through a focused beam. A generating functional is developed to fully describe the experimental physical system as well as the statistics. Histograms of the measured time delay between photon counts are fit by an analytical solution describing the static as well as diffusing regimes. To determine empirical fitting parameters, fluorescence correlation spectroscopy is used in parallel to the photon counting. For expedient analysis, we find that the distribution's deviation from a single Poisson shows a difference between two single fluor monomers or a double fluor aggregate of the same total intensities. Initial studies were performed on fixed-state aggregates limited to dimerization. However preliminary results on reactive species suggest that the method can be used to characterize any aggregating system.

Biophysics↗
PubMed2004

Thermal force approach to molecular evolution.

Recent experiments are discussed where temperature gradients across mesoscopic pores are shown to provide essential mechanisms for autonomous molecular evolution. On the one hand, laminar thermal convection can drive DNA replication as the molecules are continuously cycled between hot and cold regions of a chamber. On the other hand, thermophoresis can accumulate charged biopolymers in similar convection settings. The experiments show that temperature differences analogous to those across porous rocks present a robust nonequilibrium boundary condition to feed the replication and accumulation of evolving molecules. It is speculated that similar nonequilibrium conditions near porous submarine hydrothermal mounds could have triggered the origin of life. In such a scenario, the encapsulation of cells with membranes would be a later development. It is expected that detailed studies of mesoscopic boundary conditions under nonequilibrium conditions will reveal new connecting pieces in the fascinating puzzle of the origins of life.

Biophysical Phenomena↗
PubMed2003

Principles of cell-free genetic circuit assembly.

Cell-free genetic circuit elements were constructed in a transcription-translation extract. We engineered transcriptional activation and repression cascades, in which the protein product of each stage is the input required to drive or block the following stage. Although we can find regions of linear response for single stages, cascading to subsequent stages requires working in nonlinear regimes. Substantial time delays and dramatic decreases in output production are incurred with each additional stage because of a bottleneck at the translation machinery. Faster turnover of RNA message can relieve competition between genes and stabilize output against variations in input and parameters.

Animals↗
PubMed2003

Exponential DNA replication by laminar convection.

It is shown that laminar thermal convection can drive a chain reaction of DNA replication. The convection is triggered by a constant horizontal temperature gradient, moving molecules along stationary paths between hot and cold regions. This implements the temperature cycling for the classical polymerase chain reaction (PCR). The amplification is shown to be exponential and reaches 100,000-fold gains within 25 min. Besides direct applications, the mechanism might have implications for the molecular evolution of life.

Bacteriophage lambda↗
PubMed2003

Bubble dynamics in double-stranded DNA.

We report the first measurement of the dynamics of bubble formation in double-stranded DNA. Fluctuations of fluorescence of a synthetic DNA construct, internally tagged with a fluorophore and a quencher, are monitored by fluorescence correlation spectroscopy. The relaxation dynamics follow a multistate relaxation kinetics, with a characteristic time scale of 50 microseconds. A simple model of bubble dynamics based on constant zipping-unzipping rates is proposed to account for our experimental data. The role of different secondary structures stabilizing the open bubble is tested.

DNA↗
PubMed2003

Multiplexed DNA quantification by spectroscopic shift of two microsphere cavities.

We have developed a novel, spectroscopic technique for high-sensitivity, label-free DNA quantification. We demonstrate that an optical resonance (whispering gallery mode) excited in a micron-sized silica sphere can be used to detect and measure nucleic acids. The surface of the silica sphere is chemically modified with oligonucleotides. We show that hybridization to the target DNA leads to a red shift of the optical resonance wavelength. The sensitivity of this resonant technique is measured as 6 pg/mm(2) mass loading, higher as compared to most optical single-pass devices such as surface plasmon resonance biosensors. Furthermore, we show that each microsphere can be identified by its unique resonance wavelength. Specific, multiplexed DNA detection is demonstrated by using two microspheres. The multiplexed signal from two microspheres allows us to discriminate a single nucleotide mismatch in an 11-mer oligonucleotide with a high signal-to-noise ratio of 54. This all-photonic whispering gallery mode biosensor can be integrated on a semiconductor chip that makes it an easy to manufacture, analytic component for a portable, robust lab-on-a-chip device.

Base Pair Mismatch↗
PubMed2002

In vivo imaging of quantum dots encapsulated in phospholipid micelles.

Fluorescent semiconductor nanocrystals (quantum dots) have the potential to revolutionize biological imaging, but their use has been limited by difficulties in obtaining nanocrystals that are biocompatible. To address this problem, we encapsulated individual nanocrystals in phospholipid block-copolymer micelles and demonstrated both in vitro and in vivo imaging. When conjugated to DNA, the nanocrystal-micelles acted as in vitro fluorescent probes to hybridize to specific complementary sequences. Moreover, when injected into Xenopus embryos, the nanocrystal-micelles were stable, nontoxic (<5 x 10(9) nanocrystals per cell), cell autonomous, and slow to photobleach. Nanocrystal fluorescence could be followed to the tadpole stage, allowing lineage-tracing experiments in embryogenesis.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

Trapping of DNA by thermophoretic depletion and convection.

Thermophoresis depletes DNA from a heated spot. We quantify for the first time the thermal diffusion constant D(T)=0.4x10(-8) cm(2)/s K for DNA, using fluorescent dyes and laser heating. For 5 kB DNA we extrapolate a 1000-fold depletion from a temperature difference of 50 K. Surprisingly, convection generated by the same heating can turn the depletion into trapping of DNA. Trapped DNA can form point geometries 20 microm in diameter with more than 1000-fold enhanced concentrations. The accumulation is driven only by temperature gradients and offers a new approach to biological microfluidics and replicating systems in prebiotic evolution.

Buffers↗
PubMed2002

Protein-DNA computation by stochastic assembly cascade.

The assembly of RecA on single-stranded DNA is measured and interpreted as a stochastic finite-state machine that is able to discriminate fine differences between sequences, a basic computational operation. RecA filaments efficiently scan DNA sequence through a cascade of random nucleation and disassembly events that is mechanistically similar to the dynamic instability of microtubules. This iterative cascade is a multistage kinetic proofreading process that amplifies minute differences, even a single base change. Our measurements suggest that this stochastic Turing-like machine can compute certain integral transforms.

Computational Biology↗
PubMed2002

Signal and noise in bridging PCR.

BACKGROUND: In a variant of the standard PCR reaction termed bridging, or jumping, PCR the primer-bound sequences are originally on separate template molecules. Bridging can occur if, and only if, the templates contain a region of sequence similarity. A 3' end of synthesis in one round of synthesis that terminates in this region of similarity can prime on the other. In principle, Bridging PCR (BPCR) can detect a subpopulation of one template that terminates synthesis in the region of sequence shared by the other template. This study considers the sensitivity and noise of BPCR as a quantitative assay for backbone interruptions. Bridging synthesis is also important to some methods for computing with DNA. RESULTS: In this study, BPCR was tested over a 328 base pair segment of the E. coli lac operon and a signal to noise ratio (S/N) of approximately 10 was obtained under normal PCR conditions with Taq polymerase. With special precautions in the case of Taq or by using the Stoffel fragment the S/N was improved to 100, i.e. 1 part of cut input DNA yielded the same output as 100 parts of intact input DNA. CONCLUSIONS: In the E. coli lac operator region studied here, depending on details of protocol, between 3 and 30% per kilobase of final PCR product resulted from bridging. Other systems are expected to differ in the proportion of product that is bridged consequent to PCR protocol and the sequence analyzed. In many cases physical bridging during PCR will have no informational consequence because the bridged templates are of identical sequence, but in a number of special cases bridging creates, or, destroys, information.

DNA, Bacterial↗