Synthetic Circuits for Feedback and Detection in Bacteria

Synthetic biology, by co-opting molecular machinery from existing organisms, can be used as a tool for building new genetic systems from scratch, for understanding natural networks through perturbation, or for hybrid circuits that piggy-back on existing cellular infrastructure. Although the toolbox for genetic circuits has greatly expanded in recent years, it is still difficult to separate the circuit function from its specific molecular implementation. In this thesis, we discuss the function-driven design of two synthetic circuit modules, and use mathematical models to understand the fundamental limits of circuit topology versus operating regimes as determined by the specific molecular implementation. First, we describe a protein concentration tracker circuit that sets the concentration of an output protein relative to the concentration of a reference protein. The functionality of this circuit relies on a single negative feedback loop that is implemented via small programmable protein scaffold domains. We build a mass-action model to understand the relevant timescales of the tracking behavior and how the input/output ratios and circuit gain might be tuned with circuit components. Second, we design an event detector circuit with permanent genetic memory that can record order and timing between two chemical events. This circuit was implemented using bacteriophage integrases that recombine specific segments of DNA in response to chemical inputs. We simulate expected population-level outcomes using a stochastic Markov-chain model, and investigate how inferences on past events can be made from differences between single-cell and population-level responses. Additionally, we present some preliminary investigations on spatial patterning using the event detector circuit as well as the design of stationary phase promoters for growth-phase dependent activation. These results advance our understanding of synthetic gene circuits, and contribute towards the use of circuit modules as building blocks for larger and more complex synthetic networks.

Genetically engineered sensors of cell signaling

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

Biotechnologies for cancer diagnostics : cell sorting, protein analysis and imaging of cellular metabolism

This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called “Nucleic Acid Cell Sorting (NACS)”, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.

Secondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called “Radiopharmaceutical Imaging Chip” (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.

The last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.

Radiobiological and biochemical investigations of polyoma virus-cell interactions

The cytolytic interaction of Polyoma virus with mouse embryo cells has been studied by radiobiological methods known to distinguish temperate from virulent bacteriophage. No evidence for "temperate" properties of Polyoma was found. During the course of these studies, it was observed that the curve of inactivation of Polyoma virus by ultraviolet light had two components - a more sensitive one at low doses, and a less sensitive one at higher doses. Virus which survives a low dose has an eclipse period similar to that of unirradiated virus, while virus surviving higher doses shows a significantly longer eclipse period. If Puromycin is present during the early part of the eclipse period, the survival curve becomes a single exponential with the sensitivity of the less sensitive component. These results suggest a repair mechanism in mouse cells which operates more effectively if virus development is delayed.

A comparison of the rates of inactivation of the cytolytic and transforming abilities of Polyoma by ultraviolet light, X-rays, nitrous acid treatment, or the decay of incorporated P³², showed that the transforming ability has a target size roughly 60% of that of the plaque-forming ability. It is thus concluded that only a fraction of the viral genes are necessary for causing transformation.

The appearance of virus-specific RNA in productively infected mouse kidney cells has been followed by means of hybridization between pulse-labelled RNA from the infected cells and the purified virus DNA. The results show a sharp increase in the amount of virus-specific RNA around the time of virus DNA synthesis. The presence of a small amount of virus-specific RNA in virus-free transformed cells has also been shown. This result offers strong evidence for the persistence of at least part of the viral genome in transformed cells.

Part I. Synchronization of the cell division cycle of HeLa cells in suspension cultures. Part II. Studies on chromosomal proteins of HeLa cells during the cell division cycle

Part I

These studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.

Part II

Histones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.

A detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.

Metaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.

The factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.

The relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.