Extending the type IIS toolkit for subcellular localisation in Marchantia

This project specifically aims to develop tags in Marchantia for localisation to the tonoplast (vacuole membrane), Golgi apparatus, oil bodies, peroxisome and mitochondria.

The Idea

Synthetic Biology is an emerging field that employs engineering principles for constructing genetic systems. The relative simplicity of genetic networks in Marchantia, combined with the growing set of genetic manipulation, culture, and microscopy techniques, are set to make this primitive plant a major new system for analysis and engineering (1). In order to allow for the systematic engineering of gene expression in relation to cell behaviour and metabolism in Marchantia, we need a reliable toolkit of multispectral markers as well as tags for specific subcellular locations.

The Haseloff Lab is setting up standardised practices for DNA assembly, compiling a Marchantia DNA toolkit and establishing registries to facilitate standardisation and sharing. The constructs are compatible with the OpenMTA, and thus suitable for open distribution. In the Haseoff Lab, Bernardo Pollak et al. developed Loop assembly, a simple and open type IIS assembly system (2). Susana Sauret-Gueto and team have been developing Loop-pCambia, L0 acceptor plasmids designed for Loop, and they are putting together a type IIS DNA tool kit which includes promoters, fluorescent proteins, signal peptides and other tools for synthetic biology, aiming at maximising efficiency and reproducibility of Marchantia workflows. Previous studies demonstrated that some examples of localisation sequences found in higher plants also function in Marchantia such as localisation sequences for the endoplasmic reticulum (HDEL; 3), the nucleus (SV40; N7; 3; 2) and the plasma membrane (Myr; Lti6b; 2; 3). These sequences represent useful L0 parts and are already available as Loop compatible modules in the Marchantia toolkit. Chloroplast transit peptides have also been characterised in Marchantia (4), and some of them are also available in the kit.

The aim of this project is to enlarge the Marchantia Loop toolkit with protein tags that localise to other important subcellular locations and compartments. This project specifically aims to design and test typeIIS tags for localisation to the tonoplast (vacuole membrane), golgi apparatus, oil bodies, peroxisome and mitochondria. These tools diversify the potential uses to which Marchantia can be put by the synthetic biology community, and using the open MTA Loop system, plasmids can be freely shared in between researchers.