Engineering morphogenesis of Marchantia polymorpha gemmae

Morphogenesis is an apparent yet complex process: emergence of a plant shape is the result of an intricate interplay between genetic regulation, cell physiology and mechanical processes at the tissue scale. Morphogenesis spans three levels of biological organisation, forming a nested complex system. Genes, which form the lowest level of the system, are arranged into networks that control properties of cells. Cells, which form the middle level of the system, are arranged into geometric networks, and their mechanical and chemical interactions give rise to the morphology of a whole organism. Therefore, the study of plant morphogenesis relies on understanding how genetically-driven cellular interactions influence the formation of a plant shape. Clonal propagules of Marchantia polymorpha (Marchantia), also known as gemmae, are an attractive system for studying these interactions. Gemmae are small, have a simple disk-like shape and are resilient to environmental conditions. As such, they are well-suited for fluorescent microscopy and the collection of gene expression data. These features create an opportunity to study the processes of gemma morphogenesis at tissue, cellular and genetic levels through fluorescent microscopy in a single assay. In order to enable the engineering of morphogenesis in Marchantia gemmae, tools and frameworks for obtaining, storing and analysing the observations from the three levels of biological organisation should be put into place. The work presented in this thesis focuses on the development of such tools and their application in studying the role of phytohormone auxin in Marchantia development. I introduced novel sample preparation and time-lapse imaging assays for Marchantia gemmae along with image- processing methods for the estimation of tissue expansion rates with sub-cellular resolution. These methods allowed me to hypothesise a mechanism behind the regulation of cell proliferation in Marchantia and the role auxin plays in controlling this process. Together with Bernardo Pollak, I developed MarpoDB, a gene-centric representation of the Marchantia genome that enables the search and preparation of Marchantia genetic parts for assembly into synthetic DNA constructs. I used MarpoDB to extract parts and build fluorescent reporters, providing proxies for auxin biosynthesis and signalling. The reporters were then introduced into the Marchantia genome, and the gemmae of the transgenic lines were imaged to estimate the average patterns of the reporters’ expression. The collected patterns were then overlaid on the patterns of relative tissue expansion to validate the proposed role of auxin as an inhibitor of cell proliferation and the mechanism behind its transport in Marchantia.BBSR

Discrete distributional differential expression (D3E)--a tool for gene expression analysis of single-cell RNA-seq data.

BACKGROUND: The advent of high throughput RNA-seq at the single-cell level has opened up new opportunities to elucidate the heterogeneity of gene expression. One of the most widespread applications of RNA-seq is to identify genes which are differentially expressed between two experimental conditions. RESULTS: We present a discrete, distributional method for differential gene expression (D(3)E), a novel algorithm specifically designed for single-cell RNA-seq data. We use synthetic data to evaluate D(3)E, demonstrating that it can detect changes in expression, even when the mean level remains unchanged. Since D(3)E is based on an analytically tractable stochastic model, it provides additional biological insights by quantifying biologically meaningful properties, such as the average burst size and frequency. We use D(3)E to investigate experimental data, and with the help of the underlying model, we directly test hypotheses about the driving mechanism behind changes in gene expression. CONCLUSION: Evaluation using synthetic data shows that D(3)E performs better than other methods for identifying differentially expressed genes since it is designed to take full advantage of the information available from single-cell RNA-seq experiments. Moreover, the analytical model underlying D(3)E makes it possible to gain additional biological insights

µCube: A Framework for 3D Printable Optomechanics

Scientific instruments often require the integration of mechanics, electronics and optics. While the use of 3D printing techniques and commodity electronics has lowered the cost of instrumentation, the design and prototyping of optical components and light paths can be challenging and expensive. In recent years, attempts have been made to make optical devices more affordable using 3D printing as a method for production of optomechanical components. In this paper we present an assembly standard for the production of 3D printed optical devices. We describe a framework for parametric design of modular mounts, present two modules built using the framework, and demonstrate the potential for generalised design of modular optical devices following the μCube standard.This project was supported by the Biotechnology and Biological Sciences Research Council and Engineering and Physical Sciences Research Council Synthetic Biology Research Centre supported by the Research Councils’ Synthetic Biology for Growth Programme [OpenPlant grant No. BB/L014130/1 to J.H.]; and University of Cambridge BBSRC DTP programme [M.D.

Loop assembly: a simple and open system for recursive fabrication of DNA circuits.

High efficiency methods for DNA assembly have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets or serial manipulations for the different stages of assembly. We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardised level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show construction of plasmids up to 16 genes and 38 Kb with high efficiency (>80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing. Our method provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access. This article is protected by copyright. All rights reserved.Support for the authors was provided by Becas Chile and the Cambridge Trust (to B.P.), University of Cambridge BBSRC DTP programme (to M.D.), and the Biotechnology and Biological Sciences Research Council and Engineering and Physical Sciences Research Council [OpenPlant Grant No. BB/L014130/1] (to N.P., F.F. and J.H.). Laboratory automation, nextgeneration sequencing and library construction was delivered via the BBSRC National Capability in Genomics (BB/CCG1720/1) at Earlham Institute. F.F. acknowledges funding from CONICYT Fondecyt Iniciación 11140776. F.F. and R.A.G. acknowledge funding from Fondo de Desarrollo de Areas Prioritarias (FONDAP) Center for Genome Regulation (15090007) and Millennium Nucleus Center for Plant Systems and Synthetic Biology (NC130030)

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP

Prevalence of honeybee viruses in connection with coinfection by mite Varroa destructor in region Klatovsko

Parameters for the Islam et al. data without degradation rates. Parameters for the 12,135 genes that were expressed in both cell types. (TSV 2631 kb

GeneGuard: A Modular Plasmid System Designed for Biosafety

Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin–antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided <i>in trans</i> by a specified host, whose essential <i>thyA</i> or <i>dapA</i> gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in <i>E. coli</i> and <i>B. subtilis</i> and do not disrupt the intended <i>E. coli</i> host’s growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors

The landscape of transcription factor promoter activity during vegetative development in Marchantia

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterising the transcriptional activity of TF genes in space and time is a critical step towards understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only ∼450 putative TF genes, Marchantia (Marchantia polymorpha) is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested reporter fusions for all the TF promoters in the collection and systematically analysed expression patterns in Marchantia gemmae. This allowed us to build a map of expression domains in early vegetative development and identify a set of TF-derived promoters that are active in the stem cell zone. The cell markers provide additional tools and insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that these promoter elements will be useful for cell-type-specific expression, synthetic biology applications, and functional genomics