Cell circuitry for metals: Integrative metabolism for cobalt uptake and cobalamin production
Lead Research Organisation:
Durham University
Department Name: Biosciences
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
Technical Summary
The project deals with the advancement of synthetic biology and specifically with the design, introduction and adaption of regulatory control circuits for the uptake of metal ions. In this project we will be studying how cobalt can be controlled and regulated for its incorporation into adenosylcobalamin, the biologically active form of vitamin B12. We have shown that it is possible to engineer the cobalamin pathway into E. coli but that the cobalt uptake mechanism is very inefficient with less than 0.5% incorporation of cobalt from the medium into the nutrient. The yield of cobalamin improves as the level of exogenous cobalt in the medium is increased but this then leads to inhibition of cell growth due to the toxic effect of the cobalt on Fe-S formation. To overcome this problem we will engineer into E. coli a range of specific cobalt transporters to allow for the enhanced uptake of cobalt into the cell. The concentration of cobalt within the bacterium will be monitored by use of cobalt-sensitive reporter groups, allowing the concentration of total and buffered cobalt to be determined.
A number of different components of the cobalt transport and efflux system will be characterised in molecular detail. These will inform on how they can be modified to enhance the procurement of the metal from the medium and to maintain it within the cell. A key objective is to be able to take cobalt up at much lower concentrations to allow the metal to be incorporated into cobalamin. We have also identified a protein within the cobalamin pathway, CobW, which is a cobalt-binding GTPase that may act as a metal chaperone. We will undertake a detailed characterisation of this protein and determine whether the protein is able to deliver the metal to the chelatase for its incorporation into the vitamin. Finally, we will design and integrate a cobalamin-dependent riboswitch to control the amount of cobalt that enters the cell.
A number of different components of the cobalt transport and efflux system will be characterised in molecular detail. These will inform on how they can be modified to enhance the procurement of the metal from the medium and to maintain it within the cell. A key objective is to be able to take cobalt up at much lower concentrations to allow the metal to be incorporated into cobalamin. We have also identified a protein within the cobalamin pathway, CobW, which is a cobalt-binding GTPase that may act as a metal chaperone. We will undertake a detailed characterisation of this protein and determine whether the protein is able to deliver the metal to the chelatase for its incorporation into the vitamin. Finally, we will design and integrate a cobalamin-dependent riboswitch to control the amount of cobalt that enters the cell.
Planned Impact
The research described in this application will have a major impact on several areas of science, including synthetic biology and the manipulation of metal-requiring pathways. It will permit the generation of bacterial strains into which metal uptake and utilisation can be tightly controlled through an increased understanding of the relationship between free and buffered metal. The research relates to how cells can be engineered to improve the uptake of potentially toxic metals and how the cells can be manipulated to make the metal available for biochemical pathways. This approach will be applicable to a broad range of natural products. With growing interest in secondary metabolites, such an approach is likely to prove popular with chemical biologists and medicinal chemists alike.
The research falls well within the remit of synthetic biology and is therefore addressing a key priority area. In this respect the project applies the engineering paradigm of systems design to metabolism. In essence, the project employs the re-design of existing, natural biological systems for useful purposes. The research also has the potential to engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity. The research will have application in the biomedicine and bioprocessing of pharmaceuticals and nutrients but also has the potential to be applied to the area of bioremediation.
The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. There is a current strong interest in this area and science needs to put forward a strong representation in terms of the positive contributions that it can make. The research will not only provide essential information about how pathways and enzymes can be investigated and modified, but it will also provide greater insight into the biosynthesis of cobalamin. It will demonstrate how cells can be engineered to resource their nutrient components to allow for fast and efficient synthesis. We will ensure that our findings are widely disseminated through, for example, short review articles. Furthermore, there is no doubt that the research will be of significance to those devising new strategies against disease and thus we will ensure that our findings are disseminated to those working in drug development.
The Kent and Durham groups are heavily involved in outreach programmes, through interactions with local schools and community groups. Kent is a member of the Authentic Biology Project, which is funded by a Wellcome Trust society award to bring real research into schools. Regular talks and demonstrations are given through organized events during science week and at other times by invitation via the biology4all website, ensuring there is good dissemination with the general public on a range of important issues.
The skills acquired by those involved in this project include not only a wide range of biological techniques, ranging from spectroscopy and structural biology through to microbiology and recombinant DNA technology, but could also provide the capacity to make significant contributions towards the development of biotherapeutics. The knowledge and techniques will provide those employed with skills that can be used across education and industry. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Office. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of DBIS in Durham (in agreement with collaborators in Kent), the research will be brought to the attention of many leading industrial companies.
The research falls well within the remit of synthetic biology and is therefore addressing a key priority area. In this respect the project applies the engineering paradigm of systems design to metabolism. In essence, the project employs the re-design of existing, natural biological systems for useful purposes. The research also has the potential to engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity. The research will have application in the biomedicine and bioprocessing of pharmaceuticals and nutrients but also has the potential to be applied to the area of bioremediation.
The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. There is a current strong interest in this area and science needs to put forward a strong representation in terms of the positive contributions that it can make. The research will not only provide essential information about how pathways and enzymes can be investigated and modified, but it will also provide greater insight into the biosynthesis of cobalamin. It will demonstrate how cells can be engineered to resource their nutrient components to allow for fast and efficient synthesis. We will ensure that our findings are widely disseminated through, for example, short review articles. Furthermore, there is no doubt that the research will be of significance to those devising new strategies against disease and thus we will ensure that our findings are disseminated to those working in drug development.
The Kent and Durham groups are heavily involved in outreach programmes, through interactions with local schools and community groups. Kent is a member of the Authentic Biology Project, which is funded by a Wellcome Trust society award to bring real research into schools. Regular talks and demonstrations are given through organized events during science week and at other times by invitation via the biology4all website, ensuring there is good dissemination with the general public on a range of important issues.
The skills acquired by those involved in this project include not only a wide range of biological techniques, ranging from spectroscopy and structural biology through to microbiology and recombinant DNA technology, but could also provide the capacity to make significant contributions towards the development of biotherapeutics. The knowledge and techniques will provide those employed with skills that can be used across education and industry. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Office. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of DBIS in Durham (in agreement with collaborators in Kent), the research will be brought to the attention of many leading industrial companies.
Publications
Foster AW
(2017)
A tight tunable range for Ni(II) sensing and buffering in cells.
in Nature chemical biology
Carr CE
(2017)
An XAS investigation of the nickel site structure in the transcriptional regulator InrS.
in Journal of inorganic biochemistry
Osman D
(2019)
Bacterial sensors define intracellular free energies for correct enzyme metalation.
in Nature chemical biology
Schilter D
(2019)
Finding the right match
in Nature Reviews Chemistry
Osman D
(2017)
Fine control of metal concentrations is necessary for cells to discern zinc from cobalt.
in Nature communications
Robinson NJ
(2020)
Metalation: nature's challenge in bioinorganic chemistry.
in Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
Rubio MÁ
(2015)
Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.
in Nucleic acids research
Description | This project was linked to funding to Martin Warren, Kent which led to findings related to the optimization of cobalt-containing vitamin B12 production in a heterologous host, E. coli: A strain suitable for the manufacture of vitamin B12 in industrial biotechnology. Notably, the cobalt insertion step into the nascent tetrapyrrole is limiting and this has several potential causes: One of these is being pursued in the Warren lab, and the other is that the 'tuning' of the cobalt chelatase is sub-optimal for cobalt acquisition in the heterologous E. coli host cytoplasm. We developed an approach to determine the buffered concentration of cobalt in a bacterial cell by measuring a series of thermodynamic parameters for the cobalt sensor RcnR. The approach was published in Nature Communications 2017, along with an accessible blog written for the Nature Microbiology community. This publication includes supplements that provide a detailed description of how to perform the calculations. In part, these calculations were made possible by the discovery that metal sensors are tuned to a pre-defined buffered metal concentration within a cell (published in Nature Chemical Biology, 2017). Details of how the tuning of the cobalt chelates for cobalamin (vitamin B12) production relates to the buffered intracellular cobalt concentration will be added to ResearchFish once the final two papers from this project (both currently in draft form) have been published. |
Exploitation Route | Refer to narrative impact in relation to commercial vitamin B12 production. |
Sectors | Agriculture Food and Drink Chemicals Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Cobalt containing vitamin B12 is only made by bacteria and is typically obtained from dairy products (ultimately derived from microbes in the cow rumen). We do not acquire significant amounts of B12 from our own microbiota. Thus, vegan diets need to be somehow supplemented with vitamin B12 yet this is the most expensive vitamin on the market. Therefore (along with Martin Warren, Kent) we have engaged in discussions, and developed impact-related plans (now funded via a GCRF award "enhancing cobalamin vitamin B12 bioavailability in culturally appropriate food in India"), with multiple academic and industrial partners in India (which has a high number of vegans and a prevalence of disorders related to a lack of vitamin B12) and in the UK to exploit these findings to develop more commercially viable bioprocesses for the manufacture of inexpensive vitamin B12. These plans include optimisation of the cobalt insertion step by following (at least) two distinct approaches. These approaches are informed by the findings from this award. This has led to subsequent outreach, papers and industry engagement. Vitamin B12 is also needed in commercial manufacturing processes. Discussions have also taken place (via the BBSRC Metals in Biology NIBB and again via the Warren lab) with US-UK based multinationals to enhance vitamin B12 production by exploiting key findings from this programme. This work also provided background to subsequent studies on the uptake of vitamin B12 into hydroponically grown pea seedlings, the outcomes of which have been shared with individuals working with major salad growers to produce vitamin B12 enhanced greens by indoor farming. This programme also provided a framework for jointly managing the phase 2 E3B BBSRC NIBB to promote the exploitation of "Metals in Biology" expertise via a series of collaborations between industry and academia. |
First Year Of Impact | 2017 |
Sector | Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | A calculator for metalation inside a cell (Extranet ref: OEFE3B003) |
Amount | £625,780 (GBP) |
Funding ID | BB/V006002/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2021 |
End | 09/2024 |
Title | Calculating in vivo metalation from the sensitivities of metal-sensors. |
Description | Equations, software and spreadsheets to calculate the sensitivities of metal sensors and in turn to determine metal availability inside a cell. This enables the calculation of metalation inside living cells with implications for engineering a half of the reactions of life. Includes: 1. Excel Spreadsheet (with instructions) to enable calculation of fractional DNA occupancy. 2. MATLAB codes (with instructions), to determine the buffered metal concentration from given value(s) of ?D or ?DM. 3. Supplementary equations and unique Supplementary Note 2 references in support of the above. |
Type Of Material | Technology assay or reagent |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | Too early |
URL | https://www.nature.com/articles/s41589-018-0211-4.pdf |
Title | Computational method to determine DNA occupancy by metal sensors |
Description | Computational method to determine DNA occupancy by metal sensors |
Type Of Material | Technology assay or reagent |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | Ongoing industrial collaboration. |
URL | https://www.nature.com/articles/s41467-017-02085-z#Sec23 |
Description | Interaction with industrial sponsor |
Organisation | Procter & Gamble |
Country | United States |
Sector | Private |
PI Contribution | Regular teleconference meetings (in excess of 50 over 24 months including all forms of interaction) with industrial collaborator Reciprocal exchange of materials and biologics with industrial collaborator Reciprocal visits with industrial collaborator (associated PhD students and academic staff etc) Analytical services provided for industrial partner and others, and vice versa |
Collaborator Contribution | See above |
Impact | Ongoing and confidential |
Start Year | 2012 |
Description | Metal demands during protein overexpression in bacteria |
Organisation | Biocatalysts Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | We funded and administered a Busisness Interaction voucher in a collaboration with Peter Chivers of Durham University (BIVMiB001). |
Collaborator Contribution | Protein and enzyme overexpression is a major facet of industrial biotechnology. The capacity of host organisms for protein overexpression is not naturally optimized. Transition metals are key components of the cellular machinery required for protein synthesis. The effects of protein overexpression on metal allocation within the cell have not been examined. This project will explore the effects of protein overexpression on metal utilization in Escherichia coli, a widely used platform for biologics production. The objective is to gain insight into the cellular response to the metal demands of protein overexpression. Metal allocation and utilization will be studied using RNASeq to monitor changes in gene expression that are metal--regulated or encode metal--requiring enzymes and proteins related to protein synthesis. |
Impact | Transcript levels and metal content of E. coli cells were measured at different time points post-induction during a representative fermenter run. Increased transcript levels were observed in genes important for Mg, Fe, Mn, and Ni acquisition at and beyond the midway point of overexpression (= 9 h post-induction). These increases correlated with decreases in total cellular metal content for each metal, consistent with metal deficiency sensed by metal-responsive transcriptional regulators. These deficiencies have potential effects on translational efficiency (Mg), synthesis of non-natural amino acids that affect the fidelity of tRNA charging (Ni), and posttranslational processing of newly synthesized polypeptides (Fe/Mn). No evidence for Zn-deficiency, or Cu-stress, was detected based on transcript levels and metal content. |
Start Year | 2015 |
Description | Metal utilisation in Clostridium microbial biocatalysts |
Organisation | Green Biologics |
Country | United Kingdom |
Sector | Private |
PI Contribution | We funded and administered a Business Interaction Voucher in a collaboration with Peter Chivers of Durham University (BIVMiB035) |
Collaborator Contribution | Clostridium is an exemplar for microbial fermentations that convert biomass to renewable chemicals, such as butanol (with diverse uses including consumer fuels, paints and coatings and food additives) and acetone (used in cosmetics, plastics and numerous other markets). These fermentations depend upon metalloenzymes to convert starting material to product. The optimisation of metal supply is therefore critical for cost-efficiency. Currently, little is known about the metal requirements of industrial Clostridium species, or the metal circuitry important for maintaining metal supply to these pathways. We will identify the metal requirements during different fermentative processes and in different environments (batch vs. continuous; lab vs. industrial). Results will be applied to commercial operations for immediate outcomes including reducing waste and water use impacts. To further the development of Clostridium in a variety of IB processes, the metal sensor components of the metal circuitry will be identified to enable fine-tuning of metal supply pathways. |
Impact | Project on-going |
Start Year | 2017 |
Description | EuroBIC bioinorganic chemistry conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Disseminated knowledge about the cell biology of metals which sparked questions and discussion. |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.birmingham.ac.uk/facilities/mds-cpd/conferences/eurobic/index.aspx |
Description | FASEB, Lake Tahoe, Trace Metals in Health and Disease |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Dissemination of knowledge about the cell biology of metals which sparked questions and discussion. |
Year(s) Of Engagement Activity | 2018 |
Description | Invited speaker at the 12th International Biometals web symposium, Biometals 2020. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited speaker in the opening session of an international conference (and also subsequent session chair). |
Year(s) Of Engagement Activity | 2020 |
URL | https://biometals2020.sciencesconf.org/ |
Description | Invited speaker, BBSRC NIBB BioProNET 6th annual science meeting, Manchester |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | This aim of the event was to promote collaboration between industry and academia and advertsie the opportunities arising from the E3B BBSRC NIBB. |
Year(s) Of Engagement Activity | 2019 |
URL | http://biopronetuk.org/6th-annual-science-meeting/ |
Description | Invited talk at International Conference on BioInorganic Chemistry, Interlakken, Switzerland |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | The results of our research were described which sparked questions and discussions immediately afterwards and ongoing by e-mail. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.chem.uzh.ch/dam/jcr:d809e5d0-e81b-42d0-a1c9-175c8e13e958/ICBIC19_ScientificProgram_v5.pd... |
Description | Metal Circuits, Synthetic Biology and C1 Gases Workshop |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | We organised a workshop bringing together academics and industrialists to discuss research opportunities: http://prospect.rsc.org/MiB_NIBB/metals-synthetic-biology-c1-gas-scoping-workshop/ Durham University and Green Biologics Ltd submitted a business interaction voucher (BIVMiB035) following the workshop which was funded and othere collaborations have been developed. |
Year(s) Of Engagement Activity | 2015 |
URL | https://mib-nibb.webspace.durham.ac.uk/category/outcomes/ |
Description | Metal-related antimicrobials BBSRC NIBB workshop (November 2015) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | BBSRC NIBB event with representatives from agritechnology business, consumer goods industry, pharmaceutical companies, policy and standards agencies, industrial biotechnology companies to explore the opportunities for metal-related antimicrobials (PI and RA attended). |
Year(s) Of Engagement Activity | 2015 |
Description | Metals in Biology Community Event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | We organised a workshop involving academic and industry to show case funded projects as exemplars to instigate future collaborations. |
Year(s) Of Engagement Activity | 2016 |
Description | Metals in Bioprocessing multiple BBSRC NIBB event (Hexham) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | Interaction with Bio-processing industries. PI and RA both attended and PI gave a presentation. |
Year(s) Of Engagement Activity | 2015 |
Description | NJR Bangalore |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited keynote speaker, 6th International conference on Metals in Genetics, Chemical Biology and Therapeutics, Bangalore, India. |
Year(s) Of Engagement Activity | 2016 |
Description | NJR GRC Vermont |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Dissemination of knowldege about the Cell Biology of Metals which sparked questions and discussion. |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.grc.org/cell-biology-of-metals-conference/2017/ |
Description | NJR Queen Mary University of London |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Undergraduate students |
Results and Impact | Delivered the Wills lecture. This sparked questions afterwards followed by requests for information including ICP-MS analyses (for example). |
Year(s) Of Engagement Activity | 2017 |
URL | http://www.sbcs.qmul.ac.uk/research/researchseminars/speciallectureseries/#4 |
Description | NJR San Diego ACS conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited speaker, American Chemical Society National Meeting Spring 2016, San Diego California. |
Year(s) Of Engagement Activity | 2016 |
Description | NJR TUM Munich |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Delivered lecture and interacted with postgraduate students and staff at a series of meetings. |
Year(s) Of Engagement Activity | 2017 |
Description | National Institutes of Health, Washington DC, presentation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Disseminated knowledge of the cell biology of metals which sparked questions and discussion. |
Year(s) Of Engagement Activity | 2018 |
Description | Nature Microbiology Community Blog |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited blog linked to a paper in Nature Communications |
Year(s) Of Engagement Activity | 2017 |
URL | https://naturemicrobiologycommunity.nature.com/users/71254-deenah-osman/posts/24964-sensing-the-diff... |
Description | Outreach/Press Coverage 'Veganuary' |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Quadram Institute press release used in a published media story that describes collaborative work with Durham University that was a product of multiple BBSRC funded joint programs |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.edp24.co.uk/news/health/norwich-scientists-research-on-vitamin-b12-for-vegans-8633992 |
Description | Penn State Summer Symposium in Molecular Biology |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Disseminated knowledge of the cell biology of metals which sparked questions and discussion. |
Year(s) Of Engagement Activity | 2018 |
Description | RSC Inorganic Biochemistry Discussion Group including industrial uses of metalloenzymes (York, April 2015) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Discussion of metaloenzymes and their uses (RA attended). |
Year(s) Of Engagement Activity | 2015 |
Description | Tetrapyrroles GRC, Rhode Island |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Disseminated knowledge of the cell biology of metals which sparked questions and discussion. |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.grc.org/chemistry-and-biology-of-tetrapyrroles-conference/2018/ |
Description | Utilising Rhodococcus enzymes for Industrial Biotechnology workshop (BBSRC NIBB event in York) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | Discussion about exploitation of Rhodococcus enzymes for Industrial Biotechnology (RA attended). |
Year(s) Of Engagement Activity | 2015 |