I completed my Ph.D in Biochemistry in 2016 at the University of Cambridge, under the guidance of Professor Christopher Howe and Dr Andrew Spicer of Algenuity. Together with collaborators, the Howe lab has pioneered the development of biological solar cells, which are able to produce current as a result of photosynthetic activity in cyanobacteria. I then joined Professor Patricia Harvey’s laboratory to work on the D-factory project, which aims to set up a sustainable CO2 algal biorefinary utilizing the algae Dunaliella. While there I also contributed to a European Commission report ‘food from the oceans’ as part of a high level group of scientific advisors. I have now returned to Chris Howe’s lab as part of the OpenPlant Project. The goal of this project is to create an overexpression system for transgenes that is sensitive to changed in electropotential.
Dr Zhenhua Liu
It has been estimated that plants can produce over 1 million specialized metabolites, but we know less than 0.1 % of their biosynthetic pathways. Creative methods are eminently needed to look under the iceberg of largely untapped biosynthetic pathways. As a post-doc from Anne Osbourn group at John Innes Centre, I am employing multidisciplinary approaches across bioinformatics, genetics, and chemistry, to comprehensively understand how and why plants produce this hallmark of specialized metabolites.
I am currently focusing on plants from the Brassicaceae family and systematically studying the function, evolution and biosynthesis of triterpenes from this family. I am in particular interested in pathways encoded by gene clusters. It holds great potential to mine more and novel biosynthetic pathways efficiently. However, how and why plants have evolved BGCs is still a mystery. We are aiming to gain the first understanding of their assembly, patterns of evolution and common features in a systematic fashion. This knowledge can then be used as a template guiding the research of BGCs in other types of compounds and plant families.
Dr Ingo Appelhagen
I am a molecular biologist, with a background in plant transcription factors, flavonoid biosynthesis, natural colours and metabolic engineering.
In Cathie Martin’s lab at the John Innes Centre, we have recently developed novel suspension cultures from engineered tobacco plants, to obtain stable sources of natural colourants. These cultures can produce exceptionally high levels of red to purple anthocyanin pigments, and allow a scalable constitutive year-around production under controlled conditions.
Intense blue colours are rare in nature and difficult to reproduce in pigment formulations, which is the main reason why almost all blue food colourants are synthetic dyes. Our project aims to investigate the structural properties of anthocyanin preparations that confer strong and stable blue colours and to select for anthocyanins with improved stability as reliable natural colourants. Our goal is to extend our plant cell culture approach to develop the first production platform for blue anthocyanin colourants, to replace synthetic food dyes.
Dr Gonzalo Mendoza
I obtained my PhD in Cell Biology from the University of Edinburgh, mentored by Prof. Jean D Beggs. During this time, I was interested in the spliceosome cycle, in the connection between splicing and transcription, and also in how proofreading factors help to prevent error in splicing. I spent a significant amount of time using the auxin-inducible degron to conditionally deplete essential proteins, and finding ways to improve this depletion system to get a faster and more tightly-controlled response.
My desire to embark on plant synthetic biology, while maintaining an interested in splicing and conditional expression systems, lead me to join the Plant Metabolism Group of Prof. Alison Smith in October 2017, to develop riboswitches as molecular tools to control transgene expression in algae, higher plants and other eukaryotes. The ultimate aim of this project is to develop novel inducible systems for metabolic engineering applications or as in vivo sensors of metabolites.
Dr Philip Carella
I recently completed my PhD in Dr. Robin Cameron’s lab (McMaster University, Canada), where I studied phloem-mediated long-distance immune signalling induced by a bacterial pathogen in Arabidopsis thaliana. Feeling a need to branch out a little, I joined Dr. Sebastian Schornack’s group (Sainsbury Laboratory, University of Cambridge, UK) to study interactions between filamentous microbes and non-vascular early land plants. Our goal is to identify core developmental processes required for the colonization of early land plant tissues by filamentous microbes and to understand how these processes evolved into the defense and symbiotic programs employed by higher plants. Our work will generate transcriptomics data, fluorescent marker lines and microbe inducible promoters for cell biology, and other molecular-genetic tools that will enable the OpenPlant community to explore early land plant biology.
Dr Henry Temple
Dr Bruno Martins
I am a post-doctoral researcher in James Locke's group at the Sainsbury laboratory. I am interested in how cells discriminate between different environmental states, integrate dynamic outputs from different gene circuits, and make decisions. In my current research, I use a combination of theory and time-lapse microscopy experiments to understand the dynamical coupling of the cyanobacterial circadian clock to other networks, in both endogenous and synthetic systems.
Circadian clocks are a class of networks that regulate rhythmic expression in response to daily cycles of sunlight. A large fraction of all genes in the cyanobacterium Synechococcus elongatus are clearly under circadian control. Recently, I studied the coupling of the clock to a circuit that controls expression of the gene psbAI. Genes regulated by the clock typically peak once a day, either at dawn or at dusk. However, under conditions of constant low light, I observed a doubling of the frequency of expression of psbAI, i.e., its expression peaks twice a day. Using genetic and environmental perturbations, I found these dynamics can be modulated: either single-peak or two-peak expression can be generated. Using an iteration of modelling and experiments, I then determined the network design principles underlying the dynamics of frequency doubling.
In electronics, clock signals are essential elements of complex circuits, allowing different components of the circuit to be linked and synchronised. In biology, clocks likely play a similar role. Rational designing of oscillators has been a pursuit of synthetic biology since its inception, but evolution has already endowed natural systems with extremely reliable and robust oscillators in the form of circadian clocks. If we can understand how to harness clocks to generate specific (non-circadian) frequencies, and how to systematically integrate clocks with other pathways, we could gain a powerful tool to enable the construction of more complex synthetic circuits.
Before coming to Cambridge, I did a PhD in Peter Swain's lab at the University of Edinburgh. In my PhD I used mathematical modelling to gain insight into two simple, yet ubiquitous, sensing and transductions mechanisms: allosteric sensing and phosphorylation-dephosphorylation cycles. I studied the input-output dynamics of these mechanisms in terms of the fundamental constraints inherent in their design.
Dr Lukas Müller
I’m interested in the circadian clock and its effect on physiological and agricultural performance in plants. In the OpenPlant project I am investigating the circadian clock in Marchantia polymorpha and analyze the regulation of clock behavior and outputs in this relative of early land plants. In particular, I am focusing on the primary metabolism as an excellent proxy for systemic processes and vegetative growth.
I apply fluorescent imaging tools with computational time-lapse analysis to obtain cell-specific read-outs for the whole plant in real-time. This data is intended to set the stage for both physiological engineering and systems biology approaches.
Part of my project is to engineer fluorescent proteins that are standardised and improved reporters for dynamic changes in gene expression.
Dr Eftychis Frangedakis
Eftychis did his PhD at Oxford University focusing on the evolution of developmental mechanisms in land plants. During his doctoral research he developed a strong interest and fascination for bryophytes. He then moved to the University of Tokyo to work with the least studied group of bryophytes, hornworts. After a short detour in Hong Kong he is now back to the UK working on the development of new synthetic biology tools in Marchantia.
Dr Francisco Navarro
Francisco J. Navarro's work focuses on the function of small RNA (sRNA) molecules and their use as regulatory elements in synthetic gene circuits. sRNA molecules most likely evolved as a defense mechanism against viruses and retro-transposons, and were co-opted for fine-tuning of gene expression. Their small size and predictable targeting rules make them perfect tools for regulating gene expression in synthetic gene circuits. This project is carried out in the green alga Chlamydomonas reinhardtii, which is amenable to genetic manipulation and a model organism for key plant processes, such as photosynthesis. With an sRNA pathway that resembles that of higher plants, Chlamydomonas allows the testing of proof-of-principle small RNA-based genetic devices before extrapolating to other plant species.
Francisco completed his PhD in the laboratory of Prof. Jose Manuel Siverio (University of La Laguna, Spain), studying nitrate assimilation in Hansenula polymorpha, a methylotrophic yeast with important biotechnological applications. This was followed by a postdoc in the laboratory of Sir Paul Nurse, first at The Rockefeller University, USA, and then at the London Research Institute, on cell size control and regulation of gene expression by RNA-binding proteins. Through systematic screening of a gene deletion collection of the fission yeast Schizosaccharomyces pombe, he identified a set of novel genes involved in the coordination between cell growth and cell cycle progression. In 2015, he joined the laboratory of Sir David Baulcombe in the Department of Plant Sciences, University of Cambridge.
Francisco’s research interests concern questions regarding global regulation of gene expression and limits of cell growth. These questions are relevant to synthetic biology because synthetic gene circuits are embedded into the cell’s own gene network, and so their activities are not insulated from global cell regulation. He believes that microorganisms, like Chlamydomonas, will continue to be useful research models to uncover new exciting biology, and contribute to the advancement of synthetic biology. The fast growth of unicellular algae, in addition to a range of recently developed tools and resources, are making these organisms an interesting chassis for synthetic biology, with industrial applications in the biopharming sector.
He is also a collaborator of Café Synthetique, an informal monthly meetup with public talks that brings together the Cambridge synthetic biology community
Dr Noam Chayut
I am interested in the interface between applied plant breeding and plant metabolism. In my master’s thesis we used classical breeding of passionfruit with the goal of releasing new varieties, now used by farmers. In my PhD thesis we studied carotenoid metabolism in melons and established a molecular marker now used routinely by melon breeders. More importantly, we suggested a novel non-transgenic path toward pro-vitamin A carotenoid biofortification of food crops. The objective of the current OpenPlant project is to develop pre-breeding lines of beetroot for the production of L-DOPA.
L-DOPA is used to treat Parkinson’s symptoms; however, the current costs of chemical synthesis make it unavailable for deprived populations worldwide. In addition, there is a growing demand for ‘natural’ or plant sourced pharmaceutical substances in the first world. L- DOPA, a product of tyrosine hydroxylation, is an intermediate metabolite in biosynthesis of violet and yellow betalain pigments, in Beta vulgaris (table beet). L-DOPA natural steady state levels are very low, usually undetectable. We intend to block the turnover of L-Dopa in beetroot to allow its accumulation to levels that could enable low-tech accessible production in a plant system.
Current data indicate two betalain metabolic genes that, if repressed, may boost L-Dopa accumulation. Therefore, we aim to inhibit the activity of L-DOPA-dioxygenase, and L-DOPA-cyclase in beetroot. Currently, as proof of concept, we silence both genes in hairy roots system. We adopted three complementary strategies to meet the overarching objective of L-DOPA production in beet: a) Classical genetics; b) targeted genetic mutagenesis; and c) random mutagenesis. Yellow beet, mutated in L-DOPA-cyclase exists and can be crossed with “blotchy” red beet, which probably has lower L-DOPA-dioxygenase activity. Impairing L-DOPA-dioxygenase activity in yellow beet is carried out by both the targeted mutagenesis technology CRISPR/Cas9 and the random, yet more assured, EMS mutagenesis approach.
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.
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.
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.