Biology subjects

Haseloff, J.

Publications and source records attributed to Haseloff, J..

25 records · Page 2Linked to original sources

bioRxiv2022

An optimised transformation protocol for Anthoceros agrestis and three more hornwort species

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimised transformation protocol for A. agrestis which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussi and Phaeoceros carolinianus. The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology.

plant biology↗

bioRxiv2021

Decentralizing cell-free RNA sensing with the use of low-cost cell extracts

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Here, we describe the implementation of RNA toehold switch-based sensors using E. coli cell lysate-based cell-free protein expression systems, which can be produced locally and reduce the cost of sensors by two orders of magnitude. We then demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize use of these lysates with a CRISPRi strategy to enhance the stability of linear DNAs, enabling the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

bioengineering↗

bioRxiv2020

Comparative analysis of early divergent land plants and construction of DNA tools for hyper-expression in Marchantia chloroplasts

Chloroplast genes are present at high ploidy in plants, and capable of driving very high levels of gene expression if mRNA production and stability are properly regulated. Marchantia polymorpha is a simple model plant that allows rapid transformation studies, however post-transcriptional regulation in plastids is poorly characterized in this liverwort. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 early-divergent plant species, and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilisation. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplasmic transformed plants. We have produced novel DNA tools for protein hyper-expression in a facile plant system that is a test-bed for chloroplast engineering.

plant biology↗

bioRxiv2020

Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha.

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

synthetic biology↗

bioRxiv2018

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

High efficiency methods for DNA assembly are based on sequence overlap between fragments or Type IIS restriction endonuclease cleavage and ligation. These have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets and serial manipulations for the different stages of assembly. We present Loop assembly, based on a recursive approach to DNA fabrication. Alternate use of two Type IIS restriction endonucleases and corresponding vector sets allows efficient and parallel assembly of large DNA circuits. Plasmids containing standard 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 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.

synthetic biology↗

bioRxiv2017

Programmed hierarchical patterning of bacterial populations

Modern genetic tools allow the dissection and emulation of fundamental mechanisms shaping morphogenesis in multicellular organisms. Several synthetic genetic circuits for control of multicellular patterning have been reported to date. However, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its importance in biological self-organization. We report the first synthetic genetic system implementing population-based AND logic for programmed autonomous induction of bacterial gene expression domains. We develop a ratiometric assay for bacteriophage T7 RNA polymerase activity and use it to systematically characterize different intact and split enzyme variants. We then utilize the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. We validate the AND gate-like behavior of this system both in cell suspension and in surface culture. Finally, we use the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations.\n\nAbbreviations

synthetic biology↗

bioRxiv2017

Droplet-based microfluidic analysis and screening of single plant cells

Droplet-based microfluidics has been used to facilitate high throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 protoplasts per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate that it has the potential to assess gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics.

synthetic biology↗