Bioreactor capability for the Plant and Microbe DNA Foundry

Lead Research Organisation: Earlham Institute
Department Name: Platforms and Pipelines

Abstract

Synthetic biology is the application of engineering paradigms to biological systems. Researchers are using this paradigm to design, build and test biological 'circuits' which can contain functionality taken from multiple genes or systems.

Using this methodology we are able to optimise systems for the production of bioactives - chemicals of high value, synthesised in living cells, that can be made more cost-effectively or in a more environmentally friendly fashion than with normal chemical engineering approaches.

One of the biggest challenges is when we need to test multiple aspects of a system. For instance a promoter, a unit of DNA which turns genes on or off, may work well in the root of a plant, but not well in the leaf. If we wanted to make systematic modifications to a promoter so that it can be used in a predictable fashion, we may wish to make, or synthesise, many versions of it.

The high-throughput synthetic biology laboratories at the Earlham Institute are designed to tackle this problem using automation, so that we can complete this task more quickly and more cost-effectively than if we were doing the process manually.

However, after we have made all these versions of a promoter, we will need to test its activity by taking measurements in a living system. The challenge is how we can do this in an automated and high-throughput manner.

Automated micro-bioreactors are small scale growth chambers for growing cells, and taking measurements of those cells as they grow. They can control the growing environment, by raising or reducing oxygen or carbon dioxide, they can control the nutrients in the growth chambers, and they can measure aspects of the cells as they grow - including how much oxygen they are using, how fast they grow, but critically they can also take measurements of fluorescence.

Sometimes the best way to study the performance of a small part of a cells activity is to 'tag' it with a fluorescent marker. Commonly this is GFP or 'green fluorescent protein'. This is a non-toxic way of marking the element of a cells activity. Being able to assess fluorescence in a system is a critical part of taking these high-throughput measurements.

The Earlham Institute is a leading research institute in the study of the genetics of living systems, and houses some of the UK's most advanced laboratories for these kind of studies. This project will extend our capacity and capability, in a way that enables us to share it with the entire UK research community. The Earlham Institute has been providing access to advanced biological sciences equipment for over 7 years to the UK research community through the BBSRC 'National Capability in Genomics' and prides itself on how we share our equipment, data and knowledge in transparent and open ways.

Technical Summary

High-throughput synthetic biology studies are currently bottlenecked by the ability of researchers to make rapid assessments of systems under the perturbation of a new biological part or circuit. Conversely the means to produce synthetic constructs at high volume is easily addressed through modern liquid handling automation. Phenotyping of systems can take place at many levels from single-cell to whole-plant. The use of parallel micro-bioreactors to collect phenotyping data in a fed-batch culture system enables rapid assessment of the biological activity of new parts and circuits. From measurement of enhanced or impeded growth, to the metabolic activity of a system, to the assessment of the production of fluorescent tagged proteins these systems have broad application in the synthetic biology space.

This significant addition to the Foundry capability is particularly suited to its situation on the Norwich Research Park, where it can be exploited not only for synthetic biology studies, but also for heterologous expression of high-value yeast compounds, the assessment of novel yeast bioproduction strains and the heterologous expression of effector proteins to quickly screen constructs for those creating solubilised proteins.

The system extends the capability of the Plant and Microbe DNA Foundry, set up as part of the BBSRC Synthetic Biology for Growth Programme under BB/M000966/1 "DNA Synthesis on the Norwich Research Park". The facility is focused on supporting the production of bioactives and high-value compounds from plant and microbial systems. The Foundry lab is currently administered by the staff of the BBSRC National Capability in Genomics, who are experienced in the development and deployment of platforms and technologies which are offered to the UK research community.

Planned Impact

The integration of a high-throughput micro-bioreactor, capable of assessing the performance of synthetic biology parts in multiple conditions, significantly extends the capabilities of the Plant and Microbe DNA Foundry at Earlham Institute. This added value, will accelerate synthetic biology research, reap long-term economical benefits and create new commercial opportunities in the UK.

Synthetic biology is a growing asset in the UK Bioeconomy and creates new commercial opportunities for research. The proposed capital investment promises to improve cost-effectiveness, efficiency, scalability and benefit of synthetic biology at the Earlham Institute Plant and Microbe DNA Foundry.

Rigorous assessment of constructs and methodology at an early-stage improves the success rate of scaling-up downstream. The ability to culture small volumes enables more constructs to be tested, in less time, in less space, with less reagent and energy requirements. As a consequence, a highly-effective selection process can be utilised to prevent costly and time consuming errors at the scale-up stage. Software features, such as DoE (Design of Experiments) will also help optimise environmental conditions before constructs are tested at a larger scale. DoE workflows will be supported by the bioinformatics team present in the National Capability of Genomics, who have expertise in multivariate analysis of biological systems.

Relying on multiple service providers for a project can often be unattractive due to increased transportation of valuable samples, timelines and accountable personnel. For many end-users, a DNA Foundry that can offer an end-to-end service will be an attractive, reliable and cost-saving option.

Overcoming limitations in scalability, the automated platform will attract larger projects to the UK Plant and Microbe DNA Foundry. Conversely, the DNA Foundry will also be able to manage a larger number of smaller end-to-end projects. This will increase revenue and help achieve a sustainable business model for the DNA Foundry.

Adding value to the BB/M000966/1 "DNA Synthesis at the Norwich Research Park" award, this proposed capital investment will support its aim to strengthen local and national communities through the generation of new funding opportunities, skills and jobs. The facilities at the Earlham Institute continue to strengthen the Norwich Research Park as centre for leading expertise in biotechnology and computational biology.

Publications

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Hillson N (2019) Building a global alliance of biofoundries. in Nature communications

 
Description An automated microfermentation platform (Robolector) has been acquired and installed within the Earlham Institute's DNA Foundry.

This platform allows the the growth and automated monitoring and sampling of 48 micribial cultures (e.g. bacteria, yeast).

We have established proof of concept workfloows for the growth of the bacterium, Escherichia coli. In addition we are establishing workflows for the growth and characterisation of various strains and species of yeast held within the UK National Yeast Collection and for other strains and species of bacteria used by reserachers in academia and SMEs on the Norwich Research Park. The use of this platform has been coupled with automated phenotyping experiments on other equipment in the Earlham DNA Foundry, which is enabling us to identify the genetic basis of industrially useful molecules made in microbes. This work is currently on going and we anticipate further outcomes in coming years.

In addition, scientists from across the Norwich Reserach Park (John Innes Centre, Quadrum Institute Biosciences) have engaged with the Foundry to planning experiments on this platform with the aim of
- improving methods for the production of proteins for structural studies
- production of natural products
Exploitation Route Acdemic impact is being achieved through accessibility of the platform and provision of training to the scientific community.
The platform is contributing to the sustainability of the Earlham DNA Foundry by providing a platform for the growth and analysis of the engineered organisms produced in the Foundry.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Methods developed to provide service to SME based on the Norwich Research Park.
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Title High throughput growth screening for E. coli exposed to new antimicrobials in anaerobic conditions. 
Description This method allows the phenotypic characterization of E. coli lab strains exposed to serial dilutions of antimicrobial treatments. Cells are incubated in 48 wells plates at 37 C with gentle shaking under anaerobic conditions (purged with nitrogen, with <1.0 mg/L dissolved oxygen). OD at 600nm and fluorescence can be monitored live and measured every 5 min for a duration of 24 h. Additionally pH and Dissolved Oxygen can be monitored throughout. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact This method has been developed to provide a service to a SMBs based at NRP. 
 
Title High-throughput fermentation screening for yeast with real-time monitoring of biomass, pH and dissolved oxygen 
Description The BioLector is a microfermentator able to mimic small bioreactor conditions allowing simultaneously high-throughput fermentation procedures and live monitoring of parameter such as OD, temperature, pH and fluorescence emission. Using this system, we were able to monitor five yeast strains biomass production, pH values and dissolved oxygen in real time throughout the culture duration. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact This method has been develop to increase the service porfolio of the EI DNA Foundry and phenotipically characterise yeast with industrial interest and compare strains for their ability to heterologously express a set of high-value proteins or metabolites. Provided as service at the DNA Foundry Facility 
 
Description EI DNA Foundry Tour : Nanjing Agricultural University (NAU) delegation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The developed activity was related to the presentation of the EI DNA Foundry's capabilities to a Nanjing Agricultural University (NAU) delegation. The goal of the event was to promote UK-China collaboration and the establishment of potential pioneering projects.
Year(s) Of Engagement Activity 2019
 
Description Newsletter article June 2018: Unleashing the Power of Automation for Plant and Microbial Science 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact This short communication about the DNA foundry at the Earlham had as main goal to voice up its capabilities, and inform the plant research community how to interact with us to help with their research.
Year(s) Of Engagement Activity 2018
URL https://www.garnetcommunity.org.uk/newsletters
 
Description Presentation at Automation in Synthetic Biology Workshop, University of Bristol 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk to Introduce the EI DNA Foundry to the UK SynBio community and industry/business attending the workshop.
Year(s) Of Engagement Activity 2018
URL http://www.bristol.ac.uk/brissynbio/events/2018/automation-workshop.html