Mechanistic modeling of a rewritable recombinase addressable data module

Many of the most important applications predicted to arise from Synthetic Biology will require engineered cellular memory with the capability to store data in a rewritable and reversible manner upon induction by transient stimuli. DNA recombination provides an ideal platform for cellular data storage and has allowed the development of a rewritable recombinase addressable data (RAD) module, capable of efficient data storage within a chromosome. Here, we develop the first detailed mechanistic model of DNA recombination, and validate it against a new set of in vitro data on recombination efficiencies across a range of different concentrations of integrase and gp3. Investigation of in vivo recombination dynamics using our model reveals the importance of fully accounting for all mechanistic features of DNA recombination in order to accurately predict the effect of different switching strategies on RAD module performance, and highlights its usefulness as a design tool for building future synthetic circuitry

Developments in the tools and methodologies of synthetic biology.

Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a body of knowledge from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community

Mechanistic modelling of a recombinase-based two-input temporal logic gate

Site-specific recombinases (SSRs) mediate efficient manipulation of DNA sequences in vitro and in vivo. In particular, serine integrases have been identified as highly effective tools for facilitating DNA inversion, enabling the design of genetic switches that are capable of turning the expression of a gene of interest on or off in the presence of a SSR protein. The functional scope of such circuitry can be extended to biological Boolean logic operations by incorporating two or more distinct integrase inputs. To date, mathematical modelling investigations have captured the dynamical properties of integrase logic gate systems in a purely qualitative manner, and thus such models are of limited utility as tools in the design of novel circuitry. Here, the authors develop a detailed mechanistic model of a two-input temporal logic gate circuit that can detect and encode sequences of input events. Their model demonstrates quantitative agreement with time-course data on the dynamics of the temporal logic gate, and is shown to subsequently predict dynamical responses relating to a series of induction separation intervals. The model can also be used to infer functional variations between distinct integrase inputs, and to examine the effect of reversing the roles of each integrase on logic gate output

Mechanistic Modelling of DNA Repair and Cellular Survival Following Radiation-Induced DNA Damage

Characterising and predicting the effects of ionising radiation on cells remains challenging, with the lack of robust models of the underlying mechanism of radiation responses providing a significant limitation to the development of personalised radiotherapy. In this paper we present a mechanistic model of cellular response to radiation that incorporates the kinetics of different DNA repair processes, the spatial distribution of double strand breaks and the resulting probability and severity of misrepair. This model enables predictions to be made of a range of key biological endpoints (DNA repair kinetics, chromosome aberration and mutation formation, survival) across a range of cell types based on a set of 11 mechanistic fitting parameters that are common across all cells. Applying this model to cellular survival showed its capacity to stratify the radiosensitivity of cells based on aspects of their phenotype and experimental conditions such as cell cycle phase and plating delay (correlation between modelled and observed Mean Inactivation Doses R(2) > 0.9). By explicitly incorporating underlying mechanistic factors, this model can integrate knowledge from a wide range of biological studies to provide robust predictions and may act as a foundation for future calculations of individualised radiosensitivity

Principles for the post-GWAS functional characterisation of risk loci

Several challenges lie ahead in assigning functionality to susceptibility SNPs. For example, most effect sizes are small relative to effects seen in monogenic diseases, with per allele odds ratios usually ranging from 1.15 to 1.3. It is unclear whether current molecular biology methods have enough resolution to differentiate such small effects. Our objective here is therefore to provide a set of recommendations to optimize the allocation of effort and resources in order maximize the chances of elucidating the functional contribution of specific loci to the disease phenotype. It has been estimated that 88% of currently identified disease-associated SNP are intronic or intergenic. Thus, in this paper we will focus our attention on the analysis of non-coding variants and outline a hierarchical approach for post-GWAS functional studies

Taming a Potent Mutator: Identification and Characterization of Novel Mechanisms of Regulating Antibody Diversification in B-Lymphocytes

Often the needs of an organism exceed the number of genes in the genome. Thus, modification of the genes, themselves, or of the gene products is necessary. This becomes particularly important in cells of the immune system, which have to combat a virtually infinite array of foreign pathogens. Blymphocytes, the mediators of humoral immunity, have developed extensive mechanisms of gene diversification collectively known as antibody diversification. Antibody diversification, a set of processes necessary for an organism to mount a specific and robust immune response, relies on Activation Induced Cytidine Deaminase (AID) to initiate two of such processes: Somatic Hypermutation (SHM) and Class Switch Recombination (CSR). AID-dependent deamination of cytidine bases within the variable region (SHM) and switch region (CSR) of the immunoglobulin locus (Ig) results in the modification of the antigen binding domain and diversity within effector function of the antibody, respectively. Though the activity of AID is known, the regulation of AID during the different stages of antibody diversification is less well understood. This question has been particularly challenging to address because of the difficulty of working with AID, which becomes insoluble when expressed in non B-cells. This thesis presents the development of a screen, which searches for interacting partners for poorly soluble proteins. This screen relies on the insolubility of the protein of interest and the ability of interacting proteins to induce solubilization via binding and masking of exposed hydrophobic domains. After validation of this screen using representative soluble and insoluble proteins, it was applied to AID and thirty putative AID binding partners were identified. A handful of these proteins were uncovered in prior interaction screens, thus underscoring the validity of this new screening approach. In addition, this thesis presents a comprehensive analysis, utilizing both in vitro and in vivo approaches, of one of the putative AID cofactors discovered in the screen, RING Finger Protein 126 (RNF126). In vitro studies revealed that RNF126 is a bona fide AID binding partner and, in addition, acts as an E3 ubiquitin ligase, modifying AID with the addition of a single ubiquitin moiety. Further, a conditional knockout model of RNF126 was generated and used to determine that RNF126 plays a role in vivo in fine-tuning AID activity during SHM and CSR. The findings presented here demonstrate the utility of a novel screening technique to search for interacting partners for insoluble proteins and, through its use, expands the list of putative AID cofactors. In addition, through a thorough analysis of a single AID binding partner, this thesis puts forth a novel mode of regulation of the potent mutating enzyme, paving the way for future research to uncover the role of mono-ubiquitinated AID during SHM and/or CSR

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for ecotoxicity.

There is growing public pressure to minimize the use of vertebrates in ecotoxicity testing; therefore, effective alternatives to toxicity tests causing suffering are being sought. This report discusses alternatives and differs in some respects from the reports of the other three groups because the primary concern is with harmful effects of chemicals at the level of population and above rather than with harmful effects upon individuals. It is concluded that progress toward the objective of minimizing testing that causes suffering would be served by the following initiatives--a clearer definition of goals and strategies when undertaking testing procedures; development of alternative assays, including in vitro test systems, that are based on new technology; development of nondestructive assays for vertebrates (e.g., biomarkers) that do not cause suffering; selection of most appropriate species, strains, and developmental stages for testing procedures (but no additional species for basic testing); better integrated and more flexible testing procedures incorporating biomarker responses, ecophysiological concepts, and ecological end points (progress in this direction depends upon expert judgment). In general, testing procedures could be made more realistic, taking into account problems with mixtures, and with volatile or insoluble chemicals

The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

Heat shock protein 90 (HSP90) is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies.NVP-AUY922 potently radiosensitized cells in vitro at low nanomolar concentrations with a concurrent depletion of radioresistance-linked client proteins. Radiosensitization by NVP-AUY922 was verified for the first time in vivo in a human head and neck squamous cell carcinoma xenograft model in athymic mice, as measured by delayed tumor growth and increased surrogate end-point survival (p = <0.0001). NVP-AUY922 was shown to ubiquitously inhibit resolution of dsDNA damage repair correlating to delayed Rad51 foci formation in all cell lines tested. Additionally, NVP-AUY922 induced a stalled mitotic phenotype, in a cell line-dependent manner, in HeLa and HN5 cell lines irrespective of radiation exposure. Cell cycle analysis indicated that NVP-AUY922 induced aberrant mitotic entry in all cell lines tested in the presence of radiation-induced DNA damage due to ubiquitous CHK1 depletion, but resultant downstream cell cycle effects were cell line dependent.These results identify NVP-AUY922 as the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validate it in a clinically relevant in vivo model. Mechanistic analysis at clinically achievable concentrations demonstrated that radiosensitization is mediated by the combinatorial inhibition of cell growth and survival pathways, ubiquitous delay in Rad51-mediated homologous recombination and CHK1-mediated G(2)/M arrest, but that the contribution of cell cycle perturbation to radiosensitization may be cell line specific