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

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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.