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.
This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT3A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT3A receptors in mammalian cells.
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.
A modification of the Ishizaki et al 2008 Agrobacterium mediated Marchantia sporeling transformation protocol is used.Sterilised spores are grown for 5-7 days in ½ strength Gamborg plates prior to co-cultivation for 2 days with agrobacterium in liquid media in multiwell plates. Sporelings are then spread on media with the appropriate selective antibiotic. In about 5 days, positive transformants start to emerge.
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