No description available.

No description available.

No description available.

No description available.

No description available.

Microfluidic Antibody Capture (MAC) chips are small devices capable of quantifying biomarkers in single cells. These devices offer an all-optical approach for cell manipulation, lysis and single molecule quantification of a specific protein. This thesis details various developments to this device, both in terms of throughput and improvements to the single molecule counting process. The tumour suppressor protein p53 is a central hub for cellular stresses such as DNA damage, overproliferation and ribosomal biogenesis stress. Under stressed conditions p53 brings about the expression of a host of downstream effectors ultimately leading to DNA repair, temporary cell cycle arrest, senescence or apoptosis. The specifics of how p53 can lead to a number of different cell fate decisions are still unknown and require the development of quantitative biochemical techniques. In this thesis MAC chips are used to quantify p53 in single cells under a number of conditions. The chip data is used to create a quantitative model of p53 expression. This involved the use of stochastic simulation techniques such as the Gillespie algorithm and Approximate Bayesian Computation (ABC). These simulations determined that differences in p53 expression are best described as changes in the p53 degradation rate. This agrees with previous reports describing the p53-MDM2 relationship and its associated negative feedback loop. Lastly, attempts were made to obtain absolutely quantifiable data from the MAC chip platform. This involved calibrating the platform with known amounts of recombinant p53. By providing absolutely quantifiable data to the model of p53 expression the simulations could potentially provide real, biologically relevant parameters.

The ability of biological components to function reliably in different genetic contexts is one of the underlying fundamental principles in synthetic biology. Application of this principle allows for the development of distinct parts or bio-bricks and for the possibility of genetic abstraction. However, the sensitivity of genetic elements to changes in context can cause issues for this abstracted view of genetic elements as parts. We look at how this context sensitivity effects DNA and RNA based biosensors and we further investigate the ability of feedback to reduce noise in a biological device. Riboswitches are structural genetic regulatory RNA elements that directly couple the sensing of small molecules to gene expression. They are able to sense a wide range of small molecules and in response regulate gene expression. We investigated how changes in genetic context effected riboswitch function and how these changes affected expression from a riboswitch and adaptation of a riboswitch to a novel downstream application. To overcome these contextual difficulties with riboswitches, we have designed and introduced a novel genetic element called a ribo-attenuator. This genetic element allows for predictable tuning, insulation from contextual changes and a reduction in expression variation. Ribo-attenuators allow riboswitches to be treated as a truly modular and tunable component, and thus increase their reliability for a wide range of applications. Many biological processes use a form of feedback to achieve a highly robust and reliable output. Accurate control of a biological process is essential for critical functions in biology, from the cell cycle to proteome regulation. We design a tunable synthetic feedback network using a class of viral proteins called Integrases, which can flip the orientation of DNA segments in a digital manner. Excisionase feedback closes the loop in this system, creating a novel switch that provides a tunable and modular architecture for applications throughout synthetic biology and bio-manufacturing that require a highly robust and orthogonally controlled output. This system is highly orthogonal and evolutionary robust, and demonstrates a strong capability for regulating and reducing the variability in expression genes being transcribed under its control.

Epigenetic regulation of gene expression is critical for lineage determination during development and the stable transmission of cellular identity through cell growth and division. Chromatin modifiers are a varied group of enzymes that regulate patterns of gene expression through the modification of DNA and histones. Epigenetic modifications alter the physical structure of chromatin or recruit effectors to promote or inhibit transcription. In this study, I examined the role of pre-existing histone modifications on the activity of Polycomb and Trithorax chromatin modifying enzymes. Additionally, identification of a novel H3K36me3-reader PWWP2A may constitute a new histone crosstalk pathway between H3K36me3 and histone deacetylation. Polycomb and Trithorax proteins are two important families of transcriptional regulators that promote gene repression and activation respectively. In this study, I find that histone crosstalk between Polycomb complexes PRC1 and PRC2 promote one another's activity while inhibiting Trithorax function. Recombinant nucleosomes carrying the PRC2 modification H3K27me3 stimulates the activity of CBX-PRC1, and nucleosomes with the PRC1 modification H2AK119u1 stimulates the activity of JARID2-PRC2. H2AK119u1 negatively regulates Trithorax activity and inhibits both the H3K4 methyltransferases MLL1/4 and the H3K36 methyltransferase NSD2. Histone crosstalk appears to be an important mechanism in the regulation of the recruitment and activity of chromatin modifiers. I identified a novel H3K36me3 binding protein PWWP2A in an unbiased screen for readers of H3K27me3 and H3K36me3. Biochemical characterisation of PWWP2A in HeLa cells and mouse embryonic stem cells (mESCs) showed that PWWP2A interacts with NuRD subunits MTA1-3, HDAC1/2, and RBBP4/7. Genome-wide mapping of PWWP2A targets in mESCs shows that it is enriched over the gene bodies of highly transcribed genes sharing many gene targets with HDAC1. PWWP2A localisation correlates highly with H3K36me3, and its localisation at gene bodies is dependent on the putative H3K36me3-binding PWWP domain. In budding yeast, H3K36me3-mediated recruitment of the yeast HDAC complex Rpd3S is important for maintaining transcriptional integrity over coding regions. PWWP2A-NuRD may represents an analogous pathway of transcriptional regulation in mammalian organisms.

DNA origami offers the possibility of developing novel membrane-spanning pores with potential applications in therapeutics and in nanosensors. In this work multiscale molecular dynamics simulation approaches have been employed to understand the dynamics of a DNA nanotube, and of its interaction with lipid bilayer membranes. All-atom simulation studies performed on the DNA nanotube model allowed exploration of its conformational dynamics, and of its interactions with ions and water molecules. Simulations under different conditions (specifically force fields and temperatures) revealed consistent properties in terms of the pore lumen shape and volume, and the gating-like motions at the mouths of the central pore. Overall the DNA nanotube model has been found to be relatively soft and porous in nature. Using the conformational information obtained from these all-atom simulations, a coarse-grained model of the DNA nanotube was developed in order to study its interactions with lipid bilayer membranes on an extended (microsecond) time scale. A number of different hydrophobic anchors which stabilize the nanotube relative to the hydrophobic core of the bilayer have been explored. Local perturbation of the membrane lipids has been observed. Energetic barriers to membrane insertion and exit for DNA nanotubes have been revealed using steered molecular dynamics approaches. A stable membrane- spanning coarse-grain DNA nanopore model was converted to all-atom resolution and used as the basis of simulation to explore the effect of high salt concentration on the stability and conformational dynamics of the pore. This confirmed that the DNA nanotube was stably embedded in the bilayer, and that ions did not form an alternative permeation pathway between the pore wall and the lipids, in contrast with other recently-reported DNA nanopore designs. Overall, these studies contribute to our understanding of the conformational dynamics and membrane interactions of DNA nanopores, thus providing guidelines to design next generation DNA nanopores rendered with controlled gating properties.

The Tat and Sec pathways operate in parallel to translocate proteins across the prokaryotic cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Unlike the Sec pathway, which translocates unfolded proteins, substrates of the Tat system are transported in a folded state. In Escherichia coli, the Tat system consists of three membrane proteins, TatA, TatB and TatC. Substrates are targeted to the Tat machinery via N-terminal signal peptides that have a tripartite structure, with a polar n-region, a hydrophobic h-region and a polar c-region. They critically contain an almost invariant twin-arginine (RR) motif within the n-region that is essential to trigger Tat transport. Tat signal peptides interact with the Tat receptor complex that contains multiple copies of TatA, TatB and TatC, probably in 1:1:1 ratio. Substrate binding at the receptor drives recruitment of further TatA molecules to form an oligomer that facilitates substrate transport across the membrane by an unknown mechanism. In this study, genetic screens have been utilised to gain insight into the role of the Tat signal peptide in protein translocation. Two classes of genetic suppressors have been identified that allow normally inactive substitutions at the signal peptide RR-motif to be recognised by the Tat system, and that can also compensate for inactivating substitutions in the signal peptide binding site on TatC. The first class of the suppressors fell primarily within the transmembrane region of TatB. Biochemical analysis indicated that the suppressors did not act by restoring binding of the substituted signal peptides to the Tat machinery. Instead, they caused conformational change to the receptor complex allowing it to more readily transition to the assembled state. The second class of suppressors were isolated in the signal peptide h-region and conferred increased signal peptide hydrophobicity. These suppressors restored signal peptide binding to the Tat receptor complex. The Tat system was shown to functionally interact with highly hydrophobic signal peptides including two bona fide Sec targeting sequences. This study indicates that Tat signal peptides have dual functionality, to both target substrates to the Tat machinery and to trigger its assembly and that there is unprecedented overlap between Sec and Tat signal peptides.