Cloning the λ Switch: Digital and Markov Representations.

The lysis-lysogeny switch in E. coli due to infection from lambda phage has been extensively studied and explained by scientists of molecular biology. The bacterium either survives with the viral strand of deoxyribonucleic acid (DNA) or dies producing hundreds of viruses for propagation of infection. Many proteins transcribed after infection by λ phage take part in determining the fate of the bacterium, but two proteins that play a key role in this regard are the cI and cro dimers, which are transcribed off the viral DNA. This paper presents a novel modeling mechanism for the lysis-lysogeny switch, by transferring the interactions of the main proteins, the lambda right operator and promoter regions and the ribonucleic acid (RNA) polymerase, to a finite state machine (FSM), to determine cell fate. The FSM, and thus derived is implemented in field-programmable gate array (FPGA), and simulations have been run in random conditions. A Markov model has been created for the same mechanism. Steady state analysis has been conducted for the transition matrix of the Markov model, and the results have been generated to show the steady state probability of lysis with various model values. In this paper, it is hoped to lay down guidelines to convert biological processes into computing machines.

Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/µL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

Conformational dynamics of DNA-electrophoresis on cationic membranes.

The conformational dynamics of DNA molecules undergoing electrophoresis on a fluid substrate-supported cationic lipid bilayer is investigated using fluorescence microscopy. At low electrophoretic velocities, drift of 2-D random coils is observed. In contrast, at velocities larger than 0.3 mum/s, the DNA molecules stretch out and assume branched configurations. The cross-over scenario is explained by the observation that cationic lipids segregate underneath the adsorbed DNA and confine the DNA to its counter charge imprint on time scales shorter than the relaxation time of the imprint. The concept of a tube-like confinement of the DNA is corroborated by the observed 1/N size dependence of the electrophoretic mobility in analogy to the biased reptation model in gels. The role of membrane defects and possible applications of membrane-based electrophoresis in microfluidic devices are discussed.

Statistics of single-molecule measurements: applications in flow-cytometry sizing of DNA fragments.

BACKGROUND: The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N > 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters. METHODS: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of single-molecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM). RESULTS: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five single-molecule measurements would provide a mean value within one SD of the population mean. Additional simulations determined the number of measurements required to obtain the desired number of replicates for each subpopulation within a mixture. Application of these results to flow cytometry data for lambda/HindIII and S. aureus Mu50/SmaI DNA digests produced accurate DNA fingerprints from as few as 98 single-molecule measurements. CONCLUSIONS: A surprisingly small number of single-molecule measurements are required to obtain a mean measurement descriptive of a normally-distributed parent population.

RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations.

Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.