No abstract

Resistive pulse sensors, RPS, are allowing the transport mechanism of molecules, proteins and even nanoparticles to be characterised as they traverse pores. Previous work using RPS has shown that the size, concentration and zeta potential of the analyte can be measured. Here we use tunable resistive pulse sensing (TRPS) which utilises a tunable pore to monitor the translocation times of nanoparticles with DNA modified surfaces. We start by demonstrating that the translocation times of particles can be used to infer the zeta potential of known standards and then apply the method to measure the change in zeta potential of DNA modified particles. By measuring the translocation times of DNA modified nanoparticles as a function of packing density, length, structure, and hybridisation time, we observe a clear difference in zeta potential using both mean values, and population distributions as a function of the DNA structure. We demonstrate the ability to resolve the signals for ssDNA, dsDNA, small changes in base length for nucleotides between 15-40 bases long and even the discrimination between partial and fully complementary target sequences. Such a method has potential and applications in sensors for the monitoring of nanoparticles in both medical and environmental samples.

High-pressure liquid chromatography-tandem mass spectrometry was used to obtain a protein profile of Escherichia coli strain MG1655 grown in minimal medium with glycerol as the carbon source. By using cell lysate from only 3 ؋ 10 8 cells, at least four different tryptic peptides were detected for each of 404 proteins in a short 4-h experiment. At least one peptide with a high reliability score was detected for 986 proteins. Because membrane proteins were underrepresented, a second experiment was performed with a preparation enriched in membranes. An additional 161 proteins were detected, of which from half to two-thirds were membrane proteins. Overall, 1,147 different E. coli proteins were identified, almost 4 times as many as had been identified previously by using other tools. The protein list was compared with the transcription profile obtained on Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein products were found was detected at the mRNA level. The arithmetic mean mRNA signal intensity for these genes was 3-fold higher than that for all 4,300 protein-coding genes of E. coli. Thus, GeneChip data confirmed the high reliability of the protein list, which contains about one-fourth of the proteins of E. coli. Detection of even those membrane proteins and proteins of undefined function that are encoded by the same operons (transcriptional units) encoding proteins on the list remained low.

Mapping the landscape of possible macromolecular polymer sequences to their fitness in performing biological functions is a challenge across the biosciences. A paradigm is the case of aptamers, nucleic acids that can be selected to bind particular target molecules. We have characterized the sequence-fitness landscape for aptamers binding allophycocyanin (APC) protein via a novel Closed Loop Aptameric Directed Evolution (CLADE) approach. In contrast to the conventional SELEX methodology, selection and mutation of aptamer sequences was carried out in silico, with explicit fitness assays for 44 131 aptamers of known sequence using DNA microarrays in vitro. We capture the landscape using a predictive machine learning model linking sequence features and function and validate this model using 5500 entirely separate test sequences, which give a very high observed versus predicted correlation of 0.87. This approach reveals a complex sequence-fitness mapping, and hypotheses for the physical basis of aptameric binding; it also enables rapid design of novel aptamers with desired binding properties. We demonstrate an extension to the approach by incorporating prior knowledge into CLADE, resulting in some of the tightest binding sequences.

A way through the labyrinth: LabKC is an enzyme encoded in the biosynthesis gene cluster (lab gene cluster) of the labyrinthopeptin producer Actinomadura namibiensis. Some of its genes are homologous with those in other actinomycetes strains, for example, the model organism Streptomyces coelicolor. The functional assignment of LabKC as a kinase–cyclase suggests the formation of a new post‐translational modification by a consecutive double Michael addition (see scheme; Dha=α,β‐dehydroalanine).

Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of ϳ6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant "anaerobiome" or "proteogenome" of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO 3 ؊ and NO 2 ؊ are those encoding Pf1 bacteriophage. With anaerobic NO 2 ؊ , the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO 3 ؊ and NO 2 ؊ respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr, dnr, nar, nir, and nuo), the cysG and dksA genes were found to be required for both anaerobic NO 3 ؊ and NO 2 ؊ respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO 3 ؊ and NO 2 ؊ respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.Pseudomonas aeruginosa is a gram-negative bacterium of environmental and clinical importance that is capable of both aerobic and anaerobic respiration, the latter of which requires nitrate (NO 3 Ϫ ), nitrite (NO 2 Ϫ ), or nitrous oxide (N 2 O) as an alternative electron acceptor (24). The organism can also utilize arginine for anaerobic growth via substrate-level phosphorylation, although the final cell yield during this form of growth is abysmally low compared to that observed during anaerobic respiration (55). The most facile means to obtain anaerobic energy, however, is via respiration by NO 3 Ϫ reduction. The process of nitrate reduction can occur by two routes, the first of which is an assimilatory pathway where the nitrogen from NO 3 Ϫ is incorporated into macromolecules via formation of NH 3 . Assimilation can proceed under both aerobic and anaerobic conditions. In contrast, respiratory NO 3 Ϫ reduction (denitrification) occurs only under anaerobic conditions and involves the sequential eight-electron reduction of NO 3 Ϫ to nitrogen gas (N 2 ), with intermediates including NO 2 Ϫ , nitric oxide (NO), and N 2 O. The anaerobic process generates respiratory ...

-Aptamers are short single-stranded pieces of DNA or RNA capable of binding to analytes with specificity and high affinity. Due to their comparable selectivity, stability and cost, over the last two decades aptamers have started to challenge antibodies in their use on many technology platforms. The binding event often leads to changes in the aptamer's secondary and tertiary structure; monitoring such changes has led to the creation of many new analytical sensors. Here we demonstrate the use of a tunable resistive pulse sensing (TRPS) technology to monitor the interaction between several DNA aptamers and their target -thrombin. We immobilised the aptamers onto the surface of superparamagnetic beads, prior to their incubation with the thrombin protein. The protein binding to the aptamer caused a conformational change resulting in the shielding of the polyanion backbone; this was monitored by a change in the translocation time and pulse frequency of the particles traversing the pore. This signal was sensitive enough to allow the tagless detection of thrombin down to nanomolar levels. We further demonstrate the power of TRPS by performing real time detection and characterisation of the aptamer-target interaction and measuring the association rates of the thrombin protein to the aptamer sequences.

Properties of biological fitness landscapes are of interest to a wide sector of the life sciences, from ecology to genetics to synthetic biology. For biomolecular fitness landscapes, the information we currently possess comes primarily from two sources: sparse samples obtained from directed evolution experiments; and more fine-grained but less authentic information from 'in silico' models (such as NK-landscapes). Here we present the entire protein-binding profile of all variants of a nucleic acid oligomer 10 bases in length, which we have obtained experimentally by a series of highly parallel on-chip assays. The resulting complete landscape of sequence-binding pairs, comprising more than one million binding measurements in duplicate, has been analysed statistically using a number of metrics commonly applied to synthetic landscapes. These metrics show that the landscape is rugged, with many local optima, and that this arises from a combination of experimental variation and the natural structural properties of the oligonucleotides.