Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions

Dr. Heydrien Peyret, Prof. James Brown and OpenPlant PI Prof. George Lomonossoff published their work on improving plant transient expression through the rational design of synthetic 5' and 3' untranslated regions in expression vectors:

Peyret et al. (2019): “Fig 1: The Synth expression cassette.”

Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions

Hadrien Peyret, James K. M. Brown & George P. Lomonossoff

Plant Methods Volume 15, Article number: 108 (2019)

https://plantmethods.biomedcentral.com/articles/10.1186/s13007-019-0494-9

Abstract:

Background

The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones.

Results

In this work, we present the rational design of novel synthetic 5′ and 3′ untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ-HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5′UTRs behave as expression enhancers that outperform the HT 5′UTR present in the CPMV-HT expression system. Furthermore, we confirm the critical role played by the 3′UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV-HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ-HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs.

Conclusions

We have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications.

Two Research Fellow positions now available at the University of Manchester

The Future Biomanufacturing Research Hub is announcing Research Fellow positions:

We are pleased to announce 2 Future BRH Research fellow positions now available. Based at The University of Manchester these roles will work closely with our Spoke institutions (particularly UCL and Nottingham):

·         Position 1: Upstream Bio-Process scale up interfacing with Metabolic Engineering 

·         Position 2: Techno-economic analysis of new sustainable biomanufacturing processes

·          

The closing date for applications is Monday 7th October.  Read more and apply here.

"Plants can tell time even without a brain"

James Locke and Mark Greenwood (University of Cambridge) recently published their work on the coordination of circadian rhythms between different plant organs in PLOS Biology. This research paper has now been featured in The Conversation.

The article describes how the circadian timing in different plant organs is influenced both by local organ-specific input, as well as by inter-organ communication, allowing the expression of clock proteins to move through the plant in spatial waves.

Read more about the topic using the links above.

Building a CO2-concentrating mechanism

A new blog for the PLOS Synbio Community, written by Steven Burgess (former PDRA in one of the OpenPlant labs), describes the research of Alistair McCormack and colleagues on reconstructing an algal CO2-concentrating mechanism (CCM) into higher plants.

The work is part of an international collaboration that aims to test predictions that increasing the CO2 in plant leaves, with a system adapted from algae, will enhance photosynthetic performance, and water and nutrient use efficiency.

View the blog by Steven Burgess and the paper in the Journal of Experimental Botany.

Conductive microelectrode array for potential applications in cancer tissue detection

Modern medicine has been tackling cancer-related problems for decades. Even though diagnosis and treatment methods have progressed significantly, cancer is still considered as one of the most deadly diseases in the world. Although there is no cure for cancer, detecting and treating the disease at early stages is crucial for patients’ survival. For example, when diagnosed at its earliest stage, all individuals with melanoma skin cancer survive for five years or more. When diagnosed late, only 1/4 women and 1/10 of men survive (Source: Cancer Research UK).

Our project is focusing on developing a patient-friendly device that will detect cancer at early stages. The idea is to combine an imperceptible micro-needle array with micro-electrode array technology to monitor electrical impedance of cancer tissue. Electrical impedance describes the relationship between current and voltage flowing through an electrical circuit. In simple terms it describes how well the system conducts electricity.  

A number of studies have shown the significant contrast between electrical properties of benign and malignant tissues. The contrast is due to morphological differences between normal and cancer tissue. This method shows promise for detecting cancers that may have previously gone undetected. One potential application is a biopsy needle with electrical impedance sensor array that can provide localised and accurate characterisation of biological tissue at the needle tip. Our project investigates a potential application of 3D microelectrode arrays for cancer detection.

Comparison between healthy tissue and cancerous tissue

In May 2019, we had an opportunity to take part in the OpenPlant Biomaker Challenge to develop a low-cost biological sensor. This was an excellent opportunity to try out our idea. For the first phase of the challenge, we constructed a system that contained a two-pin electrode a 2x2-electrode array integrated into a recording circuit based on components provided by Biomaker. These probes were made from subdermal needles which are commonly used for nerve monitoring or stimulation.

Experimental probe design using subdermal needles

Our detection system is powered by a DC (direct current) signal. Impedance is a concept used for AC (alternating current) signal and resistance is the DC equivalent. For that reason, we could only measure electrical resistance or, its inverse, electrical conductivity. After constructing the hardware, we calibrated the electrodes and tested it on phantom tissue – an artificial biological systems that mimics electrical properties of normal and cancerous tissues. We were able to detect areas of increased conductivity that corresponded to phantom cancer. 

Building hardware and testing on artificial tissue (below right)

We have now started the second phase of the challenge. Thanks to the follow-on funding of £2000, we are now targeting to construct an advanced detection system. Firstly, we will incorporate industry-designed 3D microelectrode arrays and run tests on phantom tissue containing both normal and cancerous tissue. It will enable us to measure spatial distribution of conductivity in a mixed sample. Another crucial milestone will be shifting from DC to AC signal processing. Biological tissues have an additional capacitive nature due to presence of thin lipid bilayer with leaky ion-channels. A capacitor ‘blocks’ DC current but not AC current flow. Hence, impedance measurements is the conventional way for characterisation of biological tissue.

The 3D micro-electrode array from Multi Channel SystemsTM

Finally, we want to investigate if microelectrode arrays can be used to detect changes not only in tissue structure but also in neuronal activities. One of the characteristics of the cancer microenvironment, which drives cancer development, is the altered activity of neurons surrounding the cancer lesion. We are interested in exploring the changes in neuronal activity as a potential biomarker for early stage cancer. By monitoring neuronal activity, we could potentially improve sensitivity and specificity of the device.

The development of an accurate detection method is associated with a number of challenges. Background noise, sensitivity, false positives, false negatives, and specificity are the prime challenges that we need to tackle to ensure that our product can be classified as a medical device for cancer diagnosis. There is an exciting journey ahead of us. At the end, we hope to deliver a form of self-test device available to people off the shelf at their local pharmacy.

By Marta Wylot and Saksham Sharma, University of Cambridge

New publications from the Baulcombe lab

OpenPlant PI David Baulcombe and colleagues recently published two papers: (1) on the miRNA-Argonaute machinery in the unicellular green alga Chlamydomonas reinhardtii, and (2) on the application of miRNAs for regulation of synthetic gene systems in this organism:

Chung et al. (2019): “Figure 1: Structural features of Chlamydomonas Argonautes.”

Distinct roles of Argonaute in the green alga Chlamydomonas reveal evolutionary conserved mode of miRNA-mediated gene expression

Betty Y.-W. Chung, Adrian Valli, Michael J. Deery, Francisco J. Navarro, Katherine Brown, Silvia Hnatova, Julie Howard, Attila Molnar & David C. Baulcombe

Sci Rep. 2019; 9: 11091. doi: 10.1038/s41598-019-47415-x

https://www.nature.com/articles/s41598-019-47415-x.pdf

Abstract:

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3′UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas, and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identifed as the predominant miRNA with efects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.

Navarro and Baulcombe (2019): “Figure 1: Construction of a synthetic circuit to measure miRNA-dependent gene repression.”

miRNA-mediated regulation of synthetic gene circuits in the green alga Chlamydomonas reinhardtii

Francisco J. Navarro and David C. Baulcombe

ACS Synth Biol. 2019 February 15; 8(2): 358–370. doi:10.1021/acssynbio.8b00393.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396871/pdf/emss-81902.pdf

Abstract:

microRNAs (miRNAs), small RNA molecules of 20–24 nts, have many features that make them useful tools for gene expression regulation — small size, flexible design, target predictability and action at a late stage of the gene expression pipeline. In addition, their role in fine-tuning gene expression can be harnessed to increase robustness of synthetic gene networks. In this work we apply a synthetic biology approach to characterize miRNA-mediated gene expression regulation in the unicellular green alga Chlamydomonas reinhardtii. This characterization is then used to build tools based on miRNAs, such as synthetic miRNAs, miRNA-responsive 3’UTRs, miRNA decoys and self-regulatory loops. These tools will facilitate the engineering of gene expression for new applications and improved traits in this alga.

Genome-wide transcription factor binding in leaves from C3 and C4 grasses

OpenPlant PI Julian Hibberd and colleagues published their work on the transcription factor binding repertoire associated with C3 and C4 photosynthesis:

Burgess et al. (2019): “Fig 6: Hyper-conserved cis-elements in grasses recruited into C4 photosynthesis.”

Genome-wide transcription factor binding in leaves from C3 and C4 grasses

Steven J Burgess, Ivan Reyna-Llorens, Sean Ross Stevenson, Pallavi Singh, Katja Jaeger, and Julian M Hibberd

Plant Cell. 2019. pii: tpc.00078.2019. doi: 10.1105/tpc.19.00078.

http://www.plantcell.org/content/plantcell/early/2019/08/19/tpc.19.00078.full.pdf

Abstract:

The majority of plants use C3 photosynthesis, but over sixty independent lineages of angiosperms have evolved the C4 pathway. In most C4 species, photosynthesis gene expression is compartmented between mesophyll and bundle sheath cells. We performed DNaseI-SEQ to identify genome-wide profiles of transcription factor binding in leaves of the C4 grasses Zea mays, Sorghum bicolor and Setaria italica as well as C3 Brachypodium distachyon. In C4 species, while bundle sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that impact gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C4 photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C4 photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general.

New LinkedIn group for synthetic biology centres: The Synbio Network

The Synbio Centres and the Foundries in the UK

In this Linked-In group present and former members of the UK Synthetic Biology Research Centres, the DNA foundries and the Synbio Innovation and Knowledge Centre can connect with each other. Through this group we aim to maintain and build the network of synbio scientists in and outside the UK. This provides us with a forum through which we can stay in touch, make new connections, stay up to date on developments in the sector, exchange ideas, and build new collaborations. Please join our group and spread the word among your colleagues!

https://www.linkedin.com/groups/13754621/

OpenPlant Forum 2019: Smart Design for the Future of Bio-economy

The last OpenPlant forum took place last month at Murray Edwards College, Cambridge. With this year’s focus being on “smart design for the future of bio-economy” the speakers provided insights into current, innovative and exciting research.

Bio-economy refers to all economic activity derived from biological research activity focussed on creating or improving industrial processes. The bio-economy injects a gross value of £153 billion into the British economy, generating over 4 million jobs (Office of National Statistics, 2012).

The bio-economy also presents excellent growth prospects as bioscience and biotechnology have the potential to develop new, more economically and environmentally sustainable solutions to current global challenges.

OpenPlant Biomaker showcases

Professor Jim Haselhoff (University of Cambridge) introducing the Biomaker challenge at the 2019 OpenPlant Forum.

The first day of the conference welcomed this round’s OpenPlant Biomaker teams to present their midway reports. Each interdisciplinary team has five months to design and produce either (i) low-cost instruments for biology or (ii) develop a biological resource or outreach project. Having already received an initial funding worth £1,000, teams were also given the opportunity to apply for follow-on funding of £2,000.

This year’s projects ranged from early-stage cancer detection and biophotovoltaic powered soil sensors to outreach in schools and capacity building in Africa. With each of the 25 teams presenting innovative projects, there truly was a wide range of exciting ideas to hear about.  You can read more about individual projects via the Biomaker hackster platform. Goodluck to all the teams who have applied for the follow-on funding!

OpenPlant Forum

The second day of the forum saw the official start to the conference, with Professor Jim Haselhoff introducing the OpenPlant joint initiative which promotes i) interdisciplinary exchange, ii) open technologies and iii) responsible innovation for improvement of sustainable agriculture and conservation.

Professor Susan Rosser (University of Edinburgh) and Professor George Lomonossoff (John Innes Centre) spoke in the first session of the day, presenting under the theme of mammalian and plant engineering, respectively. After a short break, Dr Leopold Parts (Wellcome Sanger Institute), Professor Jun Biao Dai (Shenzhen Institute of Advanced Technology, China) and Professor Jason Chin (MRC Laboratory of Molecular Biology) gave presentations on their work involving the use of synthetic gene systems.

The last presentations of the day came from Dr Sarah-Jane Dunn (Microsoft Research) on automated reasoning for biological networks, alongside Dr Daphne Ezer (Allan Turing Institute) and Professor Martin Howard (John Innes Centre) who also spoke under the topic of modelling and machine learning for biological systems.

Synbio start-ups panel - From left to right: Tim Brears (Evonetix), Jim Ajioka (Colorifix), Eyal Maori (Tropic Biosciences), Rob Field (Iceni Diagnostics), and Eugenio Butelli (Persephone Bio).

The day ended with a panel with representatives from the synbio start-ups Colorifix, Tropic Biosciences, Iceni Diagnostics, Persephone Bio and Evonetix, who had an interesting discussion with the audience about building and running a synthetic biology start-up in the present-day bioeconomy.

The final day of the forum focussed on the topics of novel approaches and technologies, and the reprogramming multicellular systems; we welcomed talks from the likes of Benedict Diederich and René Richter from the Bio-Nanoimaging group in the Leibniz Institute of Photonic Technology (Germany) on an open-source optical toolbox that can make cutting edge imaging techniques affordable and available, Dr Stephanie Mack (cancer research UK) and Professor Wendy Harwood (John Innes Centre) who spoke about genome editing techniques in mammalian and plant systems respectively, and Dr Somenath Bakshi (University of Cambridge) who informed us on his research on understanding and engineering biological networks.

With Marchantia as a model system being a hot topic for the second half of the day, Professor Mario Arteaga-Vazquez (University of Veracruz, Mexico) spoke of dicer-mediated reprogramming of cell fate specification in this system, followed by talks of  Dr Susana Sauret-Gueto and Dr Eftychis Frangedakis (University of Cambridge) who updated us on the advances in the Marchantia research in the Haseloff Lab.

The final evening of the OpenPlant Forum 2019

Throughout the day we were updated on the recent research activities in the OpenPlant centre by exciting talks by post-doc and PhD students from various different research groups. A wide range of topics were covered, including: vaccine development, insect pheromone production, plant metabolite biosynthesis, transient expression systems, cell wall engineering, plant immune responses, and transcriptional regulation in cyanobacteria.

With the final talk of the conference delivered by Roger Castells-Graells (John Innes Centre) on virus maturation, the last OpenPlant conference came to an end. We would like to thank all those who attended as well as those who made the conference possible and worked diligently behind the scenes to make such an exciting conference happen.

Integrated Genomic and Transcriptomic Analysis of the Peridinin Dinoflagellate Amphidinium carterae Plastid

OpenPlant PI Chris Howe and colleagues published their work on control of plastid gene expression in the dinoflagellate Amphidinium carterae:

Integrated Genomic and Transcriptomic Analysis of the Peridinin Dinoflagellate Amphidinium carterae Plastid

Richard G.Dorrell, R. Ellen R.Nisbet, Adrian C.Barbrook, Stephen J.L.Rowden, and Christopher J.Howe

Protist 170(4), August 2019, Pages 358-373

Abstract:

The plastid genomes of peridinin-containing dinoflagellates are highly unusual, possessing very few genes, which are located on small chromosomal elements termed “minicircles”. These minicircles may contain genes, or no recognisable coding information. Transcripts produced from minicircles may undergo unusual processing events, such as the addition of a 3' poly(U) tail. To date, little is known about the genetic or transcriptional diversity of non-coding sequences in peridinindinoflagellate plastids. These sequences include empty minicircles, and regions of non-coding DNA in coding minicircles. Here, we present an integrated plastid genome and transcriptome for the model peridinin dinoflagellate Amphidinium carterae, identifying a previously undescribed minicircle. We also profile transcripts covering non-coding regions of the psbA and petB/atpA minicircles. We present evidence that antisense transcripts are produced within the A. carterae plastid, but show that these transcripts undergo different end cleavage events from sense transcripts, and do not receive 3' poly(U) tails. The difference in processing events between sense and antisense transcripts may enable the removal of non-coding transcripts from peridinin dinoflagellate plastid transcript pools.

Build your own DNA Dave school workshop

Synthetic biology is an emerging field of research that has potential for huge impact through both societal and economic benefits. The processes and details that need to be understood to appreciate the potential for synthetic biology require a basic understanding of how genes are regulated and transcribed to make proteins and products.

DNA Dave - the DNA transcription and translation robot.

Science festivals provide an opportunity for scientists to engage with a mixed audience, that typically spend 5-10 minutes visiting individual stands. The SAW trust, together with OpenPlant, came up with the idea to build an interactive robot that could help people understand the processes involved with DNA transcription and translation; this resulted in the birth of “DNA Dave”. Dave’s head resembles the nucleus of a cell, where the double stranded DNA can be found. The DNA strands then split and one strand is transcribed into single stranded RNA that leaves the nucleus via pores, in this case represented by DNA Dave’s neck. The RNA enters the cell cytoplasm (DNA Dave’s body) and is translated by the ribosomes which produce a sequence of amino acids; this process is represented by the turning of a cog that causes a sequence of lights to flash.

Having taken the DNA Dave robot to many science festivals, it has proven to be an engaging, approachable robot, that not only helps to explain the key synthetic biology principles of DNA transcription and translation, but through doing so, enables new conversations linking key principles to scientific applications.

DNA Dave at the Cambridge Science Festival 2017

One common question that we were asked at science festivals by school teachers, was whether the DNA Dave robot was available for schools to hire. This got the ball rolling on the next DNA Dave project.

With the help of the Biomakers fund, we have set out to convert the original DNA Dave robot into a training robot and create a cross disciplinary workshop for schools, in which the schools learn to build their own DNA Dave. To achieve this, the current robot will be modified to become an effective training tool, where by all the internal workings of the robot will be accessible for hands on training. Moreover, the current micro-controller (an Arduino board) will be exchanged for a board more commonly used in schools, a micro:bit board.

DNA Dave’s current Arduino uno break out board which will be replaced by a Micro:bit board

For our launch workshop we plan on producing a workshop template, a “how to” coding and build guide, a DNA Dave CAD drawing, and materials suitable for a robot build. Students will then design and build their own DNA Dave robot using the how to guide and materials supplied, as well as receiving support and advise from us during the process.

The main principle of the workshop and robot design is to familiarise students with the process of DNA transcription and translation, but students will be given the opportunity to develop skills across coding, technical design, electronics and biology. In addition to this, all students who take part will be exposed to subjects and topics that they may not already have an interest in and therefore potentially develop an interest in a new field.

At the end of the robot builds, we hope to gather all the new DNA Dave’s to an event during the next Norwich Science Festival where schools can showcase their designs. This showcase event will also serve as an opportunity for schools to discuss their project experiences, ideas and introduce the project to new schools.

Check out our hackster profile to keep up to date with our project.

- Dr Jenni Rant, Ioannis Tamvakis and Sami Stebbings

Developer at a Bristol BioDesign Institute spin-out

Rosa Biotech is looking for a developer:

We are looking for a driven team player to support our journey of redefining biosensing and diagnostics.  Drawing upon input from our scientific research team, you will build and develop statistical/machine-learning (ML) prediction and classification algorithms for Rosa Biotech’s biosensing technology. In addition, we see broader programming and data opportunities for instance in programming its experimental robotics platforms. 

Full job description and person specification here

To apply for this role send your CV and a covering letter to hr@rosabio.tech 

Closing date is 17:00 on Friday 16th August

Transformation of the dinoflagellate chloroplasts to enable studies on coral bleaching

eLife 8:e45292 Figure 5: A chloroplast localization for chloramphenicol acetyl transferase.

Dinoflagellate algae are of enormous ecological importance as they form symbiosis with corals, providing fixed carbon to their hosts. Environmental stresses such as raised temperature lead to breakdown of the symbiosis, expulsion of the dinoflagellates, and coral bleaching. Little is known about why the symbiosis breaks down, although the generation of reactive oxygen species in the chloroplast is probably involved. Dinoflagellates have long been resistant to transformation, which has hampered research into bleaching.

With funding from the Gordon and Betty Moore Foundation, Chris Howe’s lab in the Cambridge Biochemistry Department has succeeded in transforming the chloroplast of a model dinoflagellate, Amphidinium carterae (Nimmo IC et al. (2019) Genetic transformation of the dinoflagellate chloroplast. eLife 8:e45292 DOI: https://doi.org/10.7554/eLife.45292). They exploited the highly unusual organisation of the chloroplast genome – fragmented into plasmid-like ‘minicircles’ – to make shuttle vectors for biolistic transformation. This should open the way for studies on how environmental stresses affect dinoflagellate chloroplast function and ultimately lead to coral bleaching. 

Visualising genetic circuits in space and time, with paper-based cell-free translation

We are a pair of scientists at Medical Research Council Laboratory of Molecular Biology (MRC LMB), who are passionate about helping students learn about modern science.

Synthetic biology is particularly interesting to us as we both work at the forefront of this field and appreciate how biology has transformed into more of an engineering discipline, where we learn about life by building biological systems. The same principle, i.e., learning biology by doing it, is very efficient for studying complex concepts in schools. However, performing modern synthetic biology experiments in the classroom is an expensive activity, due to the reagents, media, bacteria and lab instruments needed, not to mention the paperwork burden of dealing with genetically modified organisms.

We believe that teaching modern science can be accessible, cheap and straightforward. We are not alone in this and there are significant developments that have been done by Amino Labs, Cell-free tech and Biobits, which pursue the same goal as us: to make cutting-edge science accessible and affordable. We chose to work with the cell-free transcription-translation system (TXTL) as it is cheap to make, there is no need for safety regulations and they are highly customizable: the only thing you need is a genetic construct.

Our aim is to teach students the principles of genetic control, the foundation of synthetic biology. The first thing that struck us was the ease with which children study electric circuits by directly connecting electrical parts in chains and experimenting with them. We wanted to reiterate this logic for biology. Luckily, the major principles of genetic regulation have already been established with electrical engineering in mind; the only puzzle piece missing: to connect them physically on a breadboard.

Figure A: Cell-free transcription-translation system (TXTL) using filter paper

The TXTL is meant to be a magic mixture that produces practically any genetic part (such as Green Fluorescent Protein or T7 RNA polymerase). As a material support where the reaction is contained, we chose a filter paper. The idea was to turn these pieces of paper into functional modules by expressing proteins in them. Therefore, connecting paper pieces later will let expressed proteins move from one paper piece to another with the water flow (Fig.A).

In the end, a protein expressed in one module can affect the reaction in the other. This experimental setup simplifies studying gene circuitry, as triggers and products of the circuit are physically separated and therefore theoretically it should be easier to deal with this kind of system as opposed to a black box mixture in the tube. Also, the possibilities are practically endless as this system is highly customizable and pieces could be connected in any way that should help children to experiment with material in an unconstrained manner.

Figure B: comparison of activity between commercial mixture (Promega T7 high yield S30) and inhouse E.coli mixture.

The project started with the production of highly active TXTL E.coli mixtures. To help other laboratories that have access to only basic equipment, we used a cheap and easy protocol for preparing cell-extracts, so that our work is easily reproducible. We have prepared cell extracts either traditionally with a French press (Emulsiflex) and high-speed centrifuge, or using a cheaper and more streamlined approach by using ultrasound cell-lysis and a cooled table top centrifuge. Independent of the protocol we used for the preparation of E.coli lysate, activity was on par with the commercial mixture (Promega T7 high yield S30) (Fig.B).

Figure c: TXTL mixtures showing more active in solution than on paper.

The challenges began when we tried to run the TXTL reaction on paper: the cell-free mixtures are always active in the solution, their paper-based counterpart only gives a low signal which could only be visualized with expensive instrumentation and thus could not be used in any low-resource environments (Fig.C).

For now, we have found a viable alternative that is suitable for outreach: as opposed to lyophilizing TXTL on the paper, we freeze-dry TXTL in the tube. Surprisingly, the reaction mix was as active as the original one, and according to previous reports the reaction components retain their activity for weeks, and even months. Thus, we aim to use this ‘halfway’ TXTL product in the upcoming summer outreach. However, the battle is not over yet; we have now turned our attention to other support materials such as agarose, that does not interfere with TXTL, is cheap, could be freeze-dried and be cast in any form.

Follow the projects progress on twitter @zakir_tnimov

Project Manager (HE) GPSEP [Maternity Cover], Sainsbury Lab Cambridge

DEPARTMENT/LOCATION: Sainsbury Laboratory, Cambridge

SALARY: £36,261-£48,677

REFERENCE: PT19452

CATEGORY: Academic-related

PUBLISHED: 5 June 2019

CLOSING DATE: 30 June 2019

Applications are invited for the post of Project Manager Gatsby Plant Science Education Programme (Higher Education) in the Sainsbury Laboratory, to manage a high-profile undergraduate plant science summer school and other post-16 student engagement projects as part of a programme funded by the Gatsby Charitable Foundation.

This post offers an exciting opportunity for those with experience and interest in undergraduate education and post-16 student engagement to build on an existing, successful programme of work. The Gatsby Plant Science Summer School has demonstrable impact on some of the brightest UK biology students, and this post will manage alumni support for graduates of the summer school. The successful candidate will also have the opportunity to use their creativity and passion for plant science to devise ways of inspiring future participants in the programme and develop post-16 student plant science engagement activities.

The Gatsby Plant Science Education Programme aims to increase participation and interest in plant science in UK schools and universities, through online resources for students, school and college teachers, support for education professionals, and an annual undergraduate plant science summer school.

Applicants should have a first degree (or equivalent professional experience) in the biological sciences, preferably plant science, with a demonstrable broad knowledge of the UK Higher Education context and experience of a plant science research environment. A broad network of contacts in the plant science and/or science education communities is essential, alongside an understanding of at least one of the following fields: undergraduate bioscience education, evaluation of student engagement. Previous experience of developing partnerships on a national and local scale would be advantageous.

Successful candidates will have excellent project management skills, in addition to experience in managing financial budgets. Strong interpersonal and communication skills are required, with the ability to work in a helpful and diplomatic manner with a wide range of people at all levels.

Most importantly, we are looking for a project manager who, working with the current Summer School team will build upon the continuing success of the projects with enthusiasm.

The Laboratory provides a welcoming and collaborative environment with a wide-range of family-friendly benefits and development opportunities. More about the Sainsbury Laboratory, further information for the role and details of what the University offers to employees, can be found at: http://www.slcu.cam.ac.uk/.

Start date: The post will be available from 12 August 2019

Maternity cover: This post is fixed-term for one year or the return of the post holder, whichever is the earlier.

Applications are welcome from internal candidates who would like to apply for the role on the basis of a secondment from their current role in the University.

The interview date is Thursday 11 July 2019.

Further information: http://www.jobs.cam.ac.uk/job/21876/

Regius Professorship of Botany, Cambridge University

DEPARTMENT/LOCATION: Department of Plant Sciences

REFERENCE: PD17932

CATEGORY: Professorships/Directorships

PUBLISHED: 30 April 2019

CLOSING DATE: 28 June 2019

The Board of Electors to the Regius Professorship of Botany invite applications for this Professorship from persons whose work falls within the general field of the Professorship to take up appointment on 1 January 2020 or as soon as possible thereafter.

This appointment arises at a vibrant time for the study of plant science in Cambridge. The Department seeks to make the appointment of a scientist of outstanding calibre to this prestigious professorship who will have the opportunity to shape the direction and emphasis of plant science research, teaching and impact in Cambridge itself, and provide leadership in the subject nationally and internationally.

Candidates will have an outstanding research record of international stature in plant biology and the vision, leadership, experience and enthusiasm to build on current strengths in maintaining and developing a leading research presence. They will also have a commitment to the recruitment, training and mentoring of the next generation of researchers. They will hold a PhD or equivalent postgraduate qualification.

Standard professorial duties include teaching and research, examining, supervision and administration. The Professor will be based in Cambridge. A competitive salary will be offered.

Further information: http://www.jobs.cam.ac.uk/job/20155/

Research Associate in Plant Sciences (Fixed Term), Cambridge University

DEPARTMENT/LOCATION: Department of Plant Sciences

SALARY: £32,236-£39,609

REFERENCE: PD17760

CATEGORY: Research

PUBLISHED: 10 June 2019

CLOSING DATE: 9 July 2019

A position is open for a Leverhulme Trust-funded postdoctoral research associate based within the Department of Plant Sciences at the University of Cambridge, and supervised by Professor Beverley Glover and Professor Alex Webb.

The appointee will investigate the evolution of the WDR proteins TTG1, LWD1 and LWD2. WDR proteins form a scaffold which supports the interaction of transcription factors, allowing the regulation of diverse suites of downstream genes. Our project aims to compare TTG1 and LWD protein function and identify changes important for their functional specificity. We aim to use mutant analyses to define biological function, in combination with yeast 2-hybrid analyses to determine which proteins are involved in the interacting complexes specifying different outcomes. RNAseq and ChIPseq will be used to establish the downstream targets resulting from the activities of these protein complexes.

We are looking for a highly motivated post-doctoral scientist to work in this area. The successful candidate must be able to demonstrate a strong background in the molecular genetic analysis of Arabidopsis, including a PhD in a relevant area. Experience with some of: mutant analysis, microscopy, RNAseq and ChIPseq will be necessary. Prior experience of yeast 2-hybrid analyses and/or circadian analyses will also be an advantage.

Fixed-term: The funds for this post are available for 3 years in the first instance.

Further information: http://www.jobs.cam.ac.uk/job/19960/

Programme Manager (Earth Biogenome Project), Earlham Institute, Norwich

Salary range: £39,150 - £47,850

Post No. 1003698

Contract length: 24 months

Department: Faculty

Opening date: 04 June 2019

Closing date: 01 July 2019

Applications are invited for a Programme Manager (Earth Biogenome Project) to join the Research Faculty Office at the Earlham Institute, based in Norwich, UK.

Background:
The Earlham Institute is looking for a Programme Manager to join the new Darwin Tree of Life Programme that aims to sequence the genomes of 66,000 known species of animals, plants, protozoa and fungi in the UK. This is part of a global effort (Earth Biogenome Project) to sequence the genomes of 1.5 million species on Earth.

Work at the Earlham Institute will focus on analysing genomes to further our understanding of evolutionary processes that drive biodiversity in populations and ecosystems. We are also involved in applying genomics to the conservation and management of valuable ecosystems and to the sustainable use of biodiversity for public good.

EI is seeking a highly skilled Programme Manager to support the Institute’s involvement in the Earth Biogenome Project - a global, collaborative initiative which aims to sequence the genomes of all species of life on Earth in the next 10-20 years.

The role:
The role will be key in the application for further funding to expand our engagement in these UK and global projects. This is a diverse, vital role and an excellent opportunity for someone seeking to move away from the bench into full time project management or seeking to move to project management in this exciting area of research.

This varied and dynamic role will involve providing high quality project management for activities in the Research Faculty Office in all aspects of implementing the Institute research strategy, taking responsibility for spearheading new activities.

The ideal candidate:
To be considered for this post, applicants must possess a PhD in a relevant scientific field. Candidates should have excellent experience of managing complex research programmes/projects and a working knowledge of project management productivity tools. Financial management experience is desirable.

Candidates should have prior experience of working in an academic environment and have a good track record of scientific writing. Excellent interpersonal skills and the ability to draft scientific documents is essential for this post. Candidates should be resilient, adaptable, organised and able to work well as part of a team.

Additional information:
Salary on appointment will be within the range £39,150 to £47,850 per annum depending on qualifications and experience. This is a full time post for a contract of 2 years.

We welcome applications from candidates seeking job share, part time or alternative working patterns.

As a Disability Confident employer, we guarantee to offer an interview to all disabled applicants who meet the essential criteria for this vacancy.

Postdoctoral Research Scientist (Plant Metabolic Diversity), Earlham Institute, Norwich

Salary range: £31,250 - £38,100

Post No. 1003678

Contract length: 18 months

Department: Engineering Biology

Opening date: 04 June 2019

Closing date: 01 July 2019

Applications are invited for a Postdoctoral Research Scientist (Plant Metabolic Diversity) to join the Patron Lab at the Earlham Institute, based in Norwich, UK. In collaboration with the Osbourn Lab at the John Innes Centre, this project will be linked to the Darwin Tree of Life Project, which aims to sequence all known UK eukaryotes.

Background:
The Earlham Institute is looking for a Postdoctoral Research Scientist (Plant Metabolic Diversity) to join the new Darwin Tree of Life Programme that aims to sequence the genomes of 66,000 known species of animals, plants, protozoa and fungi in the UK.

Work at the Earlham Institute will focus on analysing genomes to further our understanding of evolutionary processes that drive biodiversity in populations and ecosystems. We are also involved in applying genomics to the conservation and management of valuable ecosystems and to the sustainable use of biodiversity for public good.

EI is seeking a Postdoctoral Research Scientist (Plant Metabolic Diversity) to support the Institute’s involvement in the Earth Biogenome Project a global, collaborative initiative which aims to sequence the genomes of 1.5 million species of life on Earth in the next 10-20 years.

The role:
This project will generate and compare genomic, transcriptomic and metabolomic datasets for a group of related plant species. The scientist will be responsible for conducting comparative analyses with the aim of exploring the genetic basis of metabolic diversity and identifying genes responsible for the presence of target metabolites.

They will work in collaboration with other scientists at the Earlham Institute and John Innes Centre to characterise candidate genes, with the eventual aim of enabling biological production of novel, high-value metabolites.

The ideal candidate:
The candidate must have a PhD in Plant Biology, Biochemistry, Bioengineering, Synthetic Biology, Evolutionary Biology, Bioinformatics or a related subject.

The project would suit either a molecular biologist or biochemist experienced in the analysis of RNA-seq/metabolomic datasets, or a bioinformatician interested in applying their expertise to understanding metabolic diversification in plants. The candidate must be motivated and interested in the application of innovative technologies to natural product biology.

Additional information:

Salary on appointment will be within the range £31,250 - £38,100 per annum depending on qualifications and experience. This is a full time post for a contract of 18 months.

As a Disability Confident employer, we guarantee to offer an interview to all disabled applicants who meet the essential criteria for this vacancy.

Postdoctoral Researcher (Osbourn Lab), John Innes Centre, Norwich

Closes: 27th June 2019 

Salary: £31,250 to £38,100 depending on qualifications and experience 

Contract: Full time until 31 March 2021.

Applications are invited for a Postdoctoral Researcher to work on a collaborative project between the laboratories of Professor Anne Osbourn (John Innes Centre) and Dr Yang Bai (Institute of Genetics and Developmental Biology, Beijing).

The successful candidate will be based at the John Innes Centre but will also visit Dr Bai’s lab at IGDB to carry out key aspects of this work relating to microbiome analysis.

This project is funded by the John Innes Centre – Chinese Academy of Sciences Centre of Excellence in Plant and Microbial Science Alliance (CEPAMS).

The role

Building on recently published work from the Osbourn and Bai labs (‘A specialized metabolic network selectively modulates Arabidopsis root microbiota’ Science 10 May 2019:Vol. 364, Issue 6440, eaau6389), the successful candidate will investigate the impact of the environment on production of host metabolites that sculpt root microbial communities. Specifically, they will:

1. Use available in silico transcriptome resources to investigate the expression of Arabidopsis biosynthetic gene clusters in roots in response to different abiotic and biotic stresses and verify the effects of different environmental conditions on gene expression experimentally by qPCR.  The impact of different environmental stresses on root microbial communities in wild type Arabidopsis will be established by root microbiome sequencing. The impact of mutation/overexpression of triterpene pathway genes on root microbiota establishment and plant fitness under different environmental conditions will then be investigated

2. Carry out in vitro tests of the effects of purified Arabidopsis root triterpenes on the growth of representative bacterial strains cultured from the Arabidopsis soil microbiota and evaluate the effects of different microbial strains on plant growth and development

3. Investigate the impact of different triterpenes (avenacins) on root microbiome establishment in oat using a suite of available thoroughly characterised avenacin pathway mutants. These experiments will reveal the role of the avenacin pathway in regulating oat root microbiota and enable comparisons to be made with findings for Arabidopsis

The ideal candidate

The post holder will work independently and ensure research and record keeping is carried out in accordance with good practice, Scientific Integrity and in compliance with local policies and any legal requirements.

The successful applicant will have a PhD in plant biology or microbiology and extensive experience of plant and/or microbial genetics and molecular biology. Experience of plant stress biology, and/or microbiome analysis are desirable.  Excellent communication and interpersonal skills are essential.

Additional information

Salary on appointment will be within the range £31,250 to £38,100 per annum depending on qualifications and experience.  This is a full time post available until 31 March 2021.

Further information and details of how to apply can be found here or contact the Human Resources team on 01603 450462 or nbi.recruitment@nbi.ac.uk  quoting reference 1003704.  Click here to find out more about working at the John Innes Centre.

We are an equal opportunities employer, actively supporting inclusivity and diversity.  As a Disability Confident organisation, we guarantee to offer an interview to all disabled applicants who meet the essential criteria for this vacancy. The John Innes Centre is also proud to hold a Gold Award from Athena SWAN and is a member of Stonewall’s Diversity Champions programme.

The closing date for applications will be 27 June 2019.  

The John Innes Centre is a registered charity (No. 223852) grant-aided by the Biotechnology and Biological Sciences Research Council and is an Equal Opportunities Employer.