OpenPlant Coordinator, University of Cambridge, United Kingdom
Ms Marta Tomaselli
I did my bachelor and master in Biotechnology in Pisa, where I discovered how fascinating plants can be. In the past, I have worked with CRISPR/Cas9 system in two different plant models: Arabidopsis thaliana and Marchantia polymorpha. These were my first experiences related to synthetic biology and they, really, got me involved into it.
In September 2016 I started as an OpenPlant PhD student at the University of Cambridge. In my first year I will do three lab rotations before beginning my final PhD project. During my first rotation in the Haseloff Lab, I have been developing microscopy techniques to image M. polymorpha gemmae. These tools will allow to retain the signal coming from fluorescent proteins in fixed samples and exploit them to achieve a 3D representation of the plant tissue.
For my second rotation, I moved to a different topic, working in the Schornack lab. This project focuses on plant-pathogen interactions: we are looking for pathogen-responsive promoters in M. polymorpha. These sequences can be exploited to generate new reporter lines.
In the future, I would like to continue working with Marchantia and exploit this plant as a model to implement new synthetic circuits. I think that the OpenPlant Community is a great resource for a PhD student, since a lot of different topics are covered by senior researchers to whom you can ask questions and suggestions about your own project.
Dr Susana Sauret-Gueto
Dr. Susana Sauret-Gueto is an experienced molecular biologist and microscopist, with a scientific background in plant growth and development.
In the OpenPlant Cambridge laboratory, she coordinates the establishment of semiautomated workflows to accelerate the generation and characterisation of genetically engineered Marchantia lines. This requires standardised practices for DNA parts building, as well as appropriate registries to facilitate sharing of resources (DNA parts and transformed plants). Susana is establishing a new facility for robotic liquid-handling around the Echo acoustic liquid handler, and an advanced microscopy facility. The microscopy hub includes a Keyence digital microscope for real-time 3D reconstruction of Marchantia plants, as well as a series of fluorescent microscopes with different resolution capabilities, for example a Leica stereo microscope with fluorescence as well as a Leica SP8 confocal microscope.
The projects being developed along these workflows aim at mapping cell and tissue types throughout Marchantia gemmae development, for basic research questions and synthetic biology approaches. The strategies include the identification of cell types by screening Enhancer Trap lines, a collection of proximal promoters from transcription factors and its screening for specific expression patterns, a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9, and the induction of localised genetic modifications through sector analysis. Susana helps managing and coordinating these interlinked projects working closely with Linda Silvestri, lab Research Technician in charge of Marchantia tissue culture, as well as with the Marchantia team of PhD and postdoc members of the lab. She is specially interested in the sector analysis project in order to dissect gene function and autonomy at the cell and tissue level.
Susana is also the main organiser of the ROC Group (Researchers with OpenPlant Cambridge), which brings together researchers in Cambridge doing Plant Synthetic Biology, both from CU and SLCU, to share common scientific interests, resources and protocols. Researchers work in a variety of plant species, but there are two core subgroups Algae-ROC and Marchantia-ROC. People are very engaged and active, which is making a difference in order to advance projects and pipelines in an efficient and collaborative way.
Mr Louis Wilson
I started as an OpenPlant PhD student at the University of Cambridge in September 2016, where I will complete three rotation projects before selecting my final PhD project. I am interested in all parts of plant biochemistry, but my projects tend to focus on the characterization and manipulation of enzymes and catalytic pathways.
In my first rotation project, I worked with Prof. Alison G Smith in Cambridge on metabolic gene clusters, developing methods for the expression of higher plant clusters in algae and yeast, and the detection of potential clusters endogenous to algae themselves. During this time I wrote a number of computer scripts for cluster detection and began the assembly of a heterologous expression system using a yeast MoClo system from the Dueber Lab.
Now in my second rotation project, I am working with Paul Dupree to study and engineer cell wall-modifying enzymes for improved crops, food and materials. I have been using OpenPlant heterologous expression systems and a transient expression construct from the Lomonossoff lab to assess the stability of glycosyltransferases in vitro, with the aim of finding better enzymes for further study and exploitation. Increasing our understanding of these enzymes may ultimately permit the creation of designer fibres and saccharides, as well as being able to manipulate the properties of plant cell walls.
Ms Linda Silvestri
As the Research Technician for the Haseloff group, I work closely with Susana Sauret-Gueto, Research Lab Manager, to ensure the smooth running of the lab. I am responsible for Marchantia polymorpha tissue culture and am working on the standardisation of existing protocols for the propagation, transformation and short and long term storage solutions, including cryopreservation.
This work will enable and facilitate the high-throughput screenings of Marchantia lines, such as the Enhancer Trap lines; a project on which several lab members collaborate. A summer student joined us for 8 weeks to work on this project and I helped with her supervision and provided laboratory training.
Dr Orr Yarkoni
I’ve been involved in Synthetic Biology for better part of the last decade. My PhD work at Newcastle University focused on facilitating bio-electronic interface via engineered pathways as part of a larger collaborative grant to create a bio-robotic hybrid device. My more recent work at the University of Cambridge was on developing a field-use whole-cell Arsenic Biosensor for deployment in South Asia (www.arsenicbiosensor.org).
I’m relatively new at working with plants and the opportunity to reengineer the Marchantia polymorpha plastid as part of the Open Plant initiative is a great point of transition into this sphere. The main focus of my contribution to Open Plant is to reconstruct the entire 121kb plastid genome in a way that makes it easier to manipulate, facilitating future work on plastid transformation in M. polymorpha and, in time, other plants. I am also working together with Haydn King from the Ajioka Lab on creating a codon optimised reporter toolkit for use in the M. polymorpha plastid, consisting of a 13 fluorescent reporters across a wide spectrum ranging from near UV to near infrared. The codon optimisation platform should also become a useful tool for future work on plastid manipulation, in Marchantia and beyond.
I worked with Jim Ajioka and Jonathan Openshaw on a science/arts collaborative project that came to be known as Syn City. The idea was to create dynamic, living sculptures using modified E. coli such that all the “paint” was living. Jonathan designed 3D printed structures of which we made moulds to cast Agar with an integrated 3D printed mesh skeleton. The modified bacteria could then be deposited on the structure, which developed colour over time. www.syncity.co.uk.
Dr Aytug Tuncel
I am applying the genome editing tools to generate novel, commercially or nutritionally valuable glucans in model crop species. The primary objective of my OpenPlant project is to generate potatoes that contain digestion-resistant starches with two major nutritional benefits: reduced calorie intake from consumption of chips, crisps and other potato-based foods and increased supply of complex carbohydrates to the microbiota of the lower gut that reduces risk of several diseases including colorectal cancer and type II diabetes.
More specifically, the project involves knocking out the gene(s) of starch branching enzymes I and/or II using crispr-CAS9 method thereby increasing the ratio of amylose to amylopectin (linear to branched starch chains) in tubers without significantly compromising the starch yield. The engineered starch will be less accessible to starch degrading enzymes, thus more resistant to digestion.
Dr Benjamin Lichman
Plants are incredible chemical factories, capable of producing a host of complex molecules that synthetic chemists struggle to produce. These compounds are produced by plants to interact with their environment, but they also have great significance for humans, as we use them for fragrances, agrichemicals and medicines. My general research interests are understanding how plants produce these valuable compounds, and how these pathways have evolved. This knowledge can then be used to produce natural products and novel chemicals in microbial or plant based platforms.
I am currently working with catnip and catmint (Nepeta cataria and N. mussinii), plants famous for their intoxicating effect on cats. The origin of this activity is the nepetalactones, a group of volatile compounds from the iridoid family of natural products. Along with their role as feline attractants, nepetalactones have also been reported to have both insect pheromone and insect repellent properties, in some cases having activities superior to DEET. The biosynthetic origin of these compounds is currently unknown. We have been using transcriptomics and proteomics to discover enzymes in the Nepeta nepetalactone biosynthesis pathway.
This work is being performed in the context of a wider chemical and genetic investigation into the mint family (Lamiaceae), a large plant family of economic importance in which Nepeta resides. I am working closely with the Mint Genome Project (funded by the NSF) to understand the evolution and regulation of natural product biosynthesis across the entire plant family. By placing newly discovered Nepeta enzymes in a detailed phylogenetic context we hope to understand the evolutionary origin of nepetalactone biosynthesis in Nepeta, and ultimately use it as a case-study for natural product evolution.
I am currently undertaking training in molecular evolution and phylogenetics with the aim of taking the principles of evolution into synthetic biology. I hope that this will reveal new methods of optimising and editing synthetic biology systems and devices.
Figure 1. Nepetalactone biosynthesis pathway in Nepeta. We are attempting to discover the enzymes that catalyse the formation of all different nepetalactone isomers. We are also attempting to understand how these enzymes have evolved. In the background is Nepeta mussinii.
Dr Hadrien Peyret
Dr Eva Thuenemann
Plants can be used as a production platform for high-value products such as vaccines, enzymes and metabolites, thereby providing a potentially fast and cost-effective alternative to other cell culture techniques. Developed within the Lomonossoff group, HyperTrans (HT) is a technology for rapid, high-level transient expression of proteins in plants. One key application of HT in the Lomonossoff group has been the production of virus-like particles for use as vaccines, scaffolds for nanotechnology and in fundamental research of virus assembly.
Virus-like particles (VLPs) consist of viral structural proteins which assemble into a particle resembling the virus but devoid of the viral genome and therefore unable to replicate. Different VLPs consisting of multiple copies of one, two or four different structural proteins have been successfully produced using the HT system and shown to be morphologically and immunologically representative of the virus. In recent years, a number of emerging diseases have been caused by enveloped viruses such as Zika virus and Chikungunya virus. Such complex virus structures can make the development of efficient vaccines and diagnostic reagents difficult and costly. In my OpenPlant project, we are working on developing strategies for the production of enveloped VLPs in plants. I am also working on modifying a large non-enveloped VLP to allow accommodation of cargo proteins on the inside of the particle.
In addition to my research project, I was involved in the planning stages for the new John Innes Centre spin-out, Leaf Systems International Ltd, which opened on the Norwich Research Park in January 2017 and will enable translation of research to indsutry through scale-up of plant-based production of proteins and metabolites.
I have also participated in various outreach activities, such as a TV interview for regional news, the Great British Bioscience Festival, JIC’s Speed Science event as well as a work experience day for school children, amongst others.
Dr Michael Stephenson
I am a chemist, with a background in natural product total synthesis, medicinal chemistry, and pharmacy. In the Osbourn group we are interested in plant secondary metabolites, and this places us at the very interface between biology and chemistry. I bring expertise in small organic molecule extraction, purification, and structural characterisation. This strengthens the group’s ability to functionally characterise biosynthetic enzymes; something which is important for many areas of research within the Osbourn lab. As such, I am involved in a number of different projects.
My main focus is on the application of transient expression in Nicotiana benthamiana towards the preparative production of high value triterpenes. I have been heavily involved in platform and method development, improving both the efficiency and scalability of procedures used within the group. I have also demonstrated the preparative utility of this platform by producing triterpenes on the gram scale.
As a medicinal chemist I am interested in applying these techniques to engineer chemical diversity, and to explore the structure activity relationships of bioactive triterpenes. I have been involved in isolating and characterising several novel triterpenes structures arising from co-expression of ‘un-natural’ combinations of biosynthetic enzymes. In addition, I have solved the structure of a number of novel and usual triterpene scaffolds, produced by oxidosqualene cyclases under investigation within the group. It would seem that despite the huge number of unique triterpene scaffolds already reported from many decades of natural product isolation, there is still a wealth of novel chemistry to be discovered, and that its discovery can be accelerated by utilising synergy between bioinformatives, synthetic biology, and chemistry.
In addition to my research, I also take a keen interest in public engagement. I have been involved in several outreach events where we attempt to present concepts in synthetic biology and chemistry in an assessable and ‘hands on’ way.
Dr Ivan Reyna-Llorens
My research involves using synthetic biology and evolution for improving agricultural traits, more specifically to improve photosynthesis. As the world population continues to expand, it is predicted that crop yields will have to increase by 50% over the next 35 years. Traditional breeding programs cannot keep pace with this current population growth rate. Plant biomass is produced by carbon dioxide (CO2) fixed by the enzyme Rubisco during photosynthesis.
This process known as C3 photosynthesis can be very inefficient as Rubisco also interacts with Oxygen (O2) in a wasteful process known as photorespiration. In order to increase yields, photorespiration should be reduced considerably. Fortunately, some plants have evolved such mechanism already. C4 photosynthesis results from a series of anatomical and biochemical modifications in the leaf that lead to photosynthesis being compartmentalized between mesophyll and bundle sheath cells. This division of labour generates a CO2 enriched environment where photorespiration is effectively abolished. C4 plants therefore produce more yield and use water and nitrogen more efficiently. The fact that C4 photosynthesis has evolved independently in more than 60 lineages allows us to think it is possible to engineer C4 photosynthesis in C3 plants. In order to engineer this trait, cell specific genetic circuits need to be developed. Unfortunately there is a limited number of genetic parts driving cell specificity in leaves. My main objective in OpenPlant is to generate a library of leaf specific motifs that can be used to drive the expression of both nuclear and plastid encoded genes in specific compartments and specific cells of leaves.
Together with colleagues in the Department of Plant Sciences, Department of Chemistry and the Department of Physics I am part of an OpenPlant fund project that aims to use microfluidics for high-throughput analysis of genetic parts. We hope to generate a whole toolbox of parts that are useful to rewire different traits.
Dr Oleg Raitskin
My project involves optimization of CRISPR/Cas9 methodology of genome editing in plants. CRISPR/Cas9 is a method of choice to perform genome engineering. There are however significant limitations which prevent broader implementation of this technology in plants.
These limitations include variable efficiency of editing at different targets, off target activity, inefficient inheritance of the created mutations, ability to edit simultaneously several targets, limited selection of targets/PAM repertoire and the need to segregate Cas9 and sgRNA from the created mutations. Numerous configurations of CRISPR/Cas9 designed to address these limitations had been published. Our aim is to establish a uniform testbed and toolkit, where many of these configurations are tested under the same conditions and their editing efficiency and off target activity will be assessed. In order to minimize variability in transgenic expression we established editing essay in plant protoplasts.
Our experimental design includes transforming protoplasts from the same harvest with different configurations of CRISPR/Cas9, including Cas9 variants which specifically edit NGG, NGAG, NGCG and NNGGGT PAMs , Cpf1s which recognise TTTN PAM, and SpCas9 variants with reduced off target activity, and assessing frequency of indels and double stranded breaks activity employing DNA capture assays and Next Generation Sequencing. Currently we gained experience in efficient extraction and transformation of the protoplasts from different plant species using our CRISPR/Cas9 constructs and we are establishing high throughput protoplast transformation methodology using automatic dispenser. In the next step we will attempt to regenerate plants from the edited protoplasts. We also trying to find the ways to perform successful CRISPR/Cas9 assisted targeted repair of gene of interest. We follow the two-step strategy: transforming the plants with “landing pad” with subsequent insertion of the repair template. Successful insertion of the repair template should restore the herbicide resistance and facilitate selection of the plants with successful repair.
I participate in the proposal for Open Plant funding titled “Establishing Low Cost Microfluidic System for Single Cell Analysis” (Dr. Steven Burgess is a principal applicant). The aim of the project is to establish cost-effective microfluidic device for single cell sorting and analysis. Significant reduction of the cost comparatively to the commercially available systems is achieved by producing some of the parts of the device such as microscope and syringe part with 3D printing technology and utilizing open source materials and repositories. Among various applications for this device will be sorting the transformed protoplasts according to the cell size and strength of the fluorescence of the transgene, and cost-effective miniaturizing and automatizing Golden Gate cloning assembly reactions.
Dr Hans-Wilhelm Nützmann
Plants produce a wide variety of specialised metabolites. These molecules play key roles in the interaction of plants with their biotic and abiotic environment. In addition to their ecological functions, plant-derived specialised metabolites are major sources of pharmaceuticals and other high-value compounds.
Recently, it was discovered that the genes for the biosynthesis of several major classes of these compounds are physically co-localised in so called ‘gene clusters’ in plant genomes. Such clustering of non-homologous genes contrasts the expected arrangement of genes in eukaryotic genomes. The co-localisation of functionally-related genes enables the formation of fundamentally different mechanisms of gene regulation in comparison to the control of dispersed genes. The purpose of this project is to improve our understanding of the transcriptional control of plant metabolic gene clusters. The focus within OpenPlant will be on chromatin related regulatory processes that govern the expression of gene clusters. By chromatin immunoprecipitation, chromosome conformation analyses and genome engineering we aim to characterise the chromatin environment at gene clusters and its impact on cluster regulation. The findings of this project will open up new opportunities for the discovery and engineering of metabolic pathways using genetic and chemical approaches. They will also underpin synthetic biology-based approaches aimed at refactoring of plant metabolic gene clusters and the development of synthetic traits.
Dr Thomas Meany
I am jointly hosted by the labs of Lisa Hall (Chemical Engineering and Biotechnology) and Jim Haseloff (Plant Science) as an interdisciplinary fellow part funded through OpenPlant. My background training is as a physicist, with a specific emphasis on optics and microfabrication. I undertook a PhD in Macquarie University (Sydney, Australia) where I developed microphotonic circuits using a 3D laser printing technique. My postdoctoral research continued in Toshiba’s Cambridge Research Labs where I worked on advanced manufacturing techniques for semiconductor quantum dots.
As a part of OpenPlant I am passionate about using optical analytical tools to study the production of secondary metabolites in specialised plant tissues. Specifically, the oil bodies of the Liverwort, Marchantia polymorpha, are potentially rich reservoirs of bio-active compounds. Using Raman microscopy, a label-free, non-destructive spectroscopy technique it is possible to study metabolic processes in real-time. As this is non-destructive it can be performed in situ and therefore both spatial and temporal information can be obtained. My hope is to correlate this data with information available using other approaches such as Matrix Assisted Laser Deposition Ionisation Mass Spectroscopy (MALDI), Gas Chromatography Mass Spectrometry (GC-MS), fluorescence microscopy and other high resolution analytical approaches. In future this could be then adapted to studies of transgenic plant species as an additional tool to study metabolic pathways. Additional model species can also be explored, for instance Nicotiana benthamiana, and potentially crop plants. I am keen to engage with teams operating in the area of natural product chemistry, metabolic engineering or teams focused on alternative analytical approaches.
Photo: Prototype microfluidic rapid 3D printed circuit fabricated during the Bio-Hackathon.
Working with the Cambridge University Technology and Enterprise club (CUTEC), I organised the UK’s first Bio-Hackathon, hosted in the Department of Plant Science (Cambridge) during the week of 21-25 June 2016. This was possible with thanks, in part, to a grant provided by the University of Cambridge Synthetic Biology Strategic Research Initiative. This event brought together a diverse interdisciplinary group of 50 participants from across the UK and the world. Teams focused on “bioware” by incorporating hardware, software and wet lab tools. One team developed a 3D printed microfluidic prototyping tool, another built a comparison software tool for DNA synthesis pricing. The winning team built a tool called “Alpha-Brick” which is a drag and drop tool for assembling bio-bricks and plugs directly into Transcriptic (a cloud laboratory) allowing immediate order of an assembled part.
Mr Bernardo Pollak
Bernardo Pollak is a 4th year PhD candidate at the University of Cambridge in Prof. Haseloff’s laboratory. As part of his PhD, he has been developing DNA assembly systems, methods for quantitative characterisation of gene expression and tools for precise manipulation of gene expression for engineering of morphogenesis in Marchantia.
Before joining the Haseloff group, he obtained his undergrad degree in Biochemistry after coursing one year of Civil Engineering in Pontificia Universidad Católica de Chile. During his undergrad thesis, he gathered support and led the first team from Chile to participate in iGEM in 2012. He has been interested in marine luminescent bacteria, isolating environmental strains and performing directed evolution experiments to obtain optimised lux reporters. As part of his luminescence work, he produced a bioluminescent dress featured in Wired as part of a collaboration with Anton Kan, former member of the Haseloff lab, and Victoria Geany from the Royal College of Arts.