CyanoSource: A foundry generated barcoded mutant library resource for the model cyanobacterium Synechocystis sp. PCC 6803

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

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Technical Summary

Our goal is to develop a much needed resource for cyanobacterial research to assist with the systematic analyses of unknown gene functions and gene regulatory networks and also for genome-wide testing of mutational effects in a common sub-strain of the model Synechocystis sp. PCC 6803 (Synechocystis). Building on the transformation and Modular Cloning (MoClo) techniques pioneered by the applicants, we will collaborate with UK DNA foundries in Norwich and Edinburgh to generate a whole genome library of gene insertion plasmids (representing 3,456 coding sequences) and produce the largest available collection of known and novel cyanobacterial mutant strains in the world.

The CyanoSource collection will contain a library of barcoded mutants for all non-essential genes that can be used for individual gene functional studies, or analysed as a pool to investigate the roles of individual genes in a given growth environment. Secondly, it will also include a unique set of conditional mutants targeting genes that are essential for survival under typical growth conditions. In the latter collection, the promoters of essential genes will be replaced seamlessly with the copper repressible promoter of the petJ gene. Thirdly, CyanoSource will make available the library of plasmids used to generate the mutants. The gene knockout plasmids will be designed with MoClo compatible cloning sites to facilitate additional targeted genome engineering applications. Lastly we will generate a publicly available dataset containing information on available plasmids and mutants that will be linked with two bioinformatic tools recently developed by the applicants, a sub-cellular proteome location map and a comparative genomics dataset, to form a single, easily accessible platform. Users will be able to identify highly conserved cyanobacterial genes, their subcellular location within Synechocystis, and order mutants and plasmids of interest.

Planned Impact

Both PIs will oversee and co-ordinate impact activities. DLS and AM have excellent experience of involvement in projects with impact (see Track Records).

Who will benefit from this resource and how?
This project provides an outstanding opportunity to integrate the skillsets of two cyanobacterial researchers with the DNA foundries at their respective institutions. Both PIs have been successful in investigating a broad range of questions related to cyanobacteria. This resource will rapidly advance their work, which will be of benefit to other researchers working in the field. This project will facilitate exchange between the foundries in Norwich and Edinburgh, leading to improvements in their bioinformatics and automation techniques. During the development of CyanoSource, the DNA foundries will also gain the opportunity to develop collaborations with other academics and industries. The PDRAs and students working on the project will benefit considerably from training at DNA foundry facilities. Thus, this project will help to train the next generation of researchers in microbiology, synthetic biology and automation. The general public will benefit from planned outreach activities. Furthermore, the availability of this resource will accelerate our understanding of the fundamental biology in oxygenic phototrophic prokaryotes and thus have wide educational value at all levels, through schools and universities.

In the short to medium term, the outputs of CyanoSource will benefit academics and researchers performing fundamental cyanobacterial research in the UK. Particularly, this work will be of interest to researchers focused on gene function studies. It will also be of considerable interest to metabolic engineers and metabolic modellers. In the medium to long term, the beneficiaries will include international academic researchers and industrial researchers. Both PIs have connections to UK companies working in the biotechnology sector (i.e. ScotBio, Cyanetics). Mutants and plasmids will be available to order for companies at similar cost to other users, while researchers in lower middle income countries will be able to obtain mutants free of charge (although they will have to cover transport costs).

How will we ensure they benefit from the resource?
Both PIs are members of Algae-UK, a Phase II BBSRC NIBB consortium of academics and industry committed to developing a UK based cyanobacterial/algal biotechnology industry. All relevant information from this project will be disseminated at Algae-UK meetings and international conferences. We will maintain an up-to-date website for CyanoSource and release a monthly newsletter (through Algae-UK) to publicise the latest plasmids and mutants produced. We will publish results in high-impact journals in a timely fashion with open access. Furthermore, we will work with our external relations teams to promote our research to a broad spectrum of end-users via press releases, contacts with journalists, and specialised news media. The use of social media tools (e.g. YouTube) will also provide a contemporary forum for knowledge exchange (see PtI). We will make use of existing contacts with PIs (see list of LoS), industry and other academics with particular research interests as soon as relevant materials are generated at CyanoSource. We will recognise and protect user confidentiality for mutants ordered, to ensure no conflicts of interest arise. We will provide mentoring to ensure uptake of PDRA training schemes, including regular progress reviews and career development plans, and participation in the promotion of CyanoSource. Results will be used as part of our regular engagement with students and non-academic audiences through outreach activities.

Publications

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Puzorjov A (2020) Phycobiliproteins from extreme environments and their potential applications. in Journal of experimental botany

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Puzorjov A (2021) Production of thermostable phycocyanin in a mesophilic cyanobacterium. in Metabolic engineering communications

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Kromdijk J (2022) Genetic variation in photosynthesis: many variants make light work. in Journal of experimental botany

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Gao H (2023) Editorial: Structure and function of chloroplasts, Volume III. in Frontiers in plant science

 
Description 2020 March. Since the start of the award (Sept 2019), we have produced one detailed review publications outlining the upcoming CyanoSource resource in Microorganisms (Gale et al. 2019 Emerging species and genome editing tools: future prospects in cyanobacterial synthetic biology. https://doi.org/10.3390/microorganisms7100409). Two further review papers are currently in the second round of review and should be accepted shortly.

2021 March.
Two new review papers have now been published in Journal of Experimental Botany (Puzorjov et al. 2020 Phycobiliproteins from extreme environments and their potential applications. https://doi.org/10.1093/jxb/eraa139) and and the most comprehensive analysis on cyanobacterial metabolism and transport available to date in Bioscience Reports (Mills et al., 2020 Current knowledge and recent advances in understanding metabolism of the model cyanobacterium Synechocystis sp. PCC 6803. https://doi.org/10.1042/BSR20193325). Further associated published research work this year included an analysis of cyanobacterial terminator efficiencies in Frontiers in Microbiology (Gale et al. 2021 Evaluation and comparison of the efficiency of transcription terminators in different cyanobacterial species. https://doi.org/10.3389/fmicb.2020.624011), and a novel method to extract phycocyanin from cyanobacteria in Algal Research (Scorza et al., 2021 Evaluation of novel 3D-printed monolithic adsorbers against conventional chromatography columns for the purification of c-phycocyanin from Spirulina. https://doi.org/10.1016/j.algal.2021.102253).

COVID has caused severe delays in project progress. Both genome foundries have had significant disruptions in workflow and consumables (e.g. availability of plasticware and molecular components), while staff on the project have been furloughed for several months (McCormick PDRA was furloughed for 4 months April - July 2020). Nevertheless, we have commenced production of plasmids (Objectives 1 and 2) and made progress with developing a high-throughput protocol for transformation in the genome foundries. In this regard, UEA, UoE, the Earlham Biofoundry and the EGF continue to have monthly meetings to discuss progress and troubleshoot challenges. To progress Objective 3, we have also made progress in developing a CyanoSource website (we've been granted our domain name cyanosource.ac.uk and are currently populating the site). To link to the site, we and have successfully secured ISSF funding to build a library for our plasmids on SynBioHub (https://synbiohub.org).

2022 March.
COVID continued to cause delays in project progress in 2021-2022. Nevertheless, we have now established a high-throughput pipeline for generating plasmids for cyanobacterial transformation at the EGF. This process is now entirely performed by the EGF, from ordering barcoded flank sequences (eBlocks), assembling these into an acceptor vector with selection markers, and verification of successful plasmid assembly (using minigels and/or nanopore sequencing). The Foundry is working through the construction of the plasmids required for Objective 1. Do to time limitations, we have modified our plans for Objective 2. We now plan to generate KO plasmids for essential genes as in Objective 1, but maintain mutants as partially segregated. Due to the design of the insertion, users will have the option to rapidly swap out the selection markers in these mutants with sequences appropriate for their experimental needs.

We have also performed a proof of concept experiment to demonstrate that the EGF equipment is able to automate the process of Synechocystis sp. PCC 6803 transformation at high-throughout (i.e. 96 transformations at once). We have requested and received a 12 month no cost extension to continue production of plasmids at the EGF, and establish the cyanobacterial transformation protocol at the EGF. We continue to have regular meetings with UEA and the Earlham Biofoundry to discuss progress and troubleshoot challenges, and the transfer of protocols between Foundries.

For objective 3, the CyanoSource website (https://cyanosource.ac.uk/) is now fully functional and contains a searchable database of all genes and available plasmids and mutants. The ISSF funded project has resulted in a library of plasmids now available on the SynBioHub design repository(https://synbiohub.org) and the generation of a biologist friendly software for updating and modifying the library. This work was recently published in Synthetic Biology (Zielinski et al. 2022, SynBio2Easy-a biologist-friendly tool for batch operations on SBOL designs with Excel inputs. https://doi.org/10.1093/synbio/ysac002). Additional research papers associated with this project have been published in Metabolic Engineering Communications (Puzorjov et al. 2021 Production of thermostable phycocyanin in a mesophilic cyanobacterium) and Bioresource Technology (Puzorjov et al. 2021 Pilot scale production, extraction and purification of a thermostable phycocyanin from Synechocystis sp. PCC 6803).

2023 March. PDRA Neninger (Edinburgh) completed her contract in 2022 July, and moved UEA with Co-I Lea-Smith to continue work on CyanoSource with the Earlham Foundry. EGF have continued to deliver verified plasmids for generating mutants via their pipeline. We currently have produced 896 plasmids targeting unique ORFs in Synechocystis sp. PCC 6803, and generated 238 mutants.
Exploitation Route CyanoSource will become a key resource for cyanobacterial researchers and algal biotechnology around the globe.
Sectors Education

Environment

Manufacturing

including Industrial Biotechology

URL http://mccormick.bio.ed.ac.uk/
 
Description A powerful directed-evolution tool for exploitation of chloroplast engineering biology
Amount £1,270,708 (GBP)
Funding ID BB/Y008162/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2024 
End 06/2026
 
Description BBSRC-Algae-UK/IBIOIC - Production scale up of a thermotolerant C-phycocyanin from a mesophilic cyanobacterial bio-platform
Amount £49,925 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2020 
End 01/2021
 
Description Development of a commercial next-generation laboratory multichamber photobioreactor
Amount £16,000 (GBP)
Organisation University of East Anglia 
Sector Academic/University
Country United Kingdom
Start 07/2021 
End 07/2022
 
Description Institutional Strategic Support Fund - Making it FAIR for sharing and reuse: developing an open access virtual repository of the CyanoSource plasmid library.
Amount £14,447 (GBP)
Organisation University of Edinburgh 
Sector Academic/University
Country United Kingdom
Start 09/2020 
End 03/2021
 
Description Rewriting The Genetic Code: The Algal Plastome As A Testbed For Basic And Applied Studies
Amount £3,146,402 (GBP)
Funding ID BB/W003538/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2022 
End 03/2027
 
Title Addgene CyanoGate Kit 
Description The CyanoGate kit contains 96 parts and acceptor vectors for generating multi-part assemblies in either integrative (suicide) or self-replicating plasmid vectors for working in cyanobacteria. The kit is compatible with the plant Modular Cloning (MoClo) syntax (also known as PhytoBricks). You require the backbone acceptor vectors in the MoClo Toolkit (Kit # 1000000044) with this kit to progress from level 0 to level T assemblies. This CyanoGate kit includes promoters, minimal (up to the transcriptional start site (TSS)) promoters, terminators, flanking sequences for HR-mediated genomic integration, selectable marker cassettes, level T (for Transformation) acceptor vectors, and parts for building CRISPRi- and sgRNA-based systems. 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? Yes  
Impact CyanoGate is now a globally available toolkit for cyanobacteria research. Addgene listed it on AddGene's "Hot Plasmids list for March 2020" (https://blog.addgene.org/hot-plasmids-march-2020). 
URL http://www.addgene.org/kits/mccormick-cyanogate/#kit-details
 
Title CyanoGate Toolkit 
Description The CyanoGate kit contains 117 parts and acceptor vectors for generating multi-part assemblies in either integrative (suicide) or self-replicating plasmid vectors for working in cyanobacteria. The core parts of the kit are supplied on a 96 well plate (from Addgene), while some additional components (i.e. 18 terminators and 3 variants of the Pcpc560 promoter) are available for separate order (from Addgene). The kit is compatible with the plant Modular Cloning (MoClo) syntax (also known as PhytoBricks). You require the backbone acceptor vectors in the MoClo Plant Parts Kit (#1000000044) (Engler et al., 2014, ACS SynBio) with this kit to progress from level 0 to level T assemblies. The CyanoGate kit includes 45 promoters, 5 minimal (up to the TSS) promoters, 21 terminators, 20 flanking sequences for HR-mediated genomic integration, 5 selectable marker cassettes, 4 level T (for Transformation) acceptor vectors, and parts for building CRISPRi- and sgRNA-based systems. 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? Yes  
Impact The kit is high-throughput Golden Gate cloning system for cyanobacterial research so will have a significant impact on this community. 
URL http://www.addgene.org/kits/mccormick-cyanogate/
 
Title CyanoSource database 
Description Website and database contains entries for 3,572 known and predicted gene coding sequences (CDs) in Synechocystis sp PCC 6803, and the presence of plasmids and/or marked mutants for each CD for community use. 
Type Of Technology Webtool/Application 
Year Produced 2022 
Open Source License? Yes  
Impact Contact from several members of the cyanobacterial research community for plasmids and Synechocystis sp PCC 6803 mutants and distribution thereof. 
URL https://cyanosource.ac.uk/
 
Description Algae UK workshop - Engagement in UK industries for algal products 
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 Workshop with companies in the UK interested in using using natural products derived from algae and cyanobacteria.
Collaborations in this workshop led to a successful Algae-UK PoC application to produce thermotolerant c-phyocyanin in Synechocystis 6803 (https://www.algae-uk.org.uk/projects/1-production-scale-up-of-a-thermotolerant-c-phycocyanin-from-a-mesophilic-cyanobacterial-bio-platform-ibioic-supported/)
Year(s) Of Engagement Activity 2019
 
Description Invited Talk (17th International Symposium on Phototrophic Prokaryotes. Liverpool, UK) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a research presentation online. Conference was attended by >100 people. Opportunity to disseminate research and advertise CyanoGate and CyanoSource.
Year(s) Of Engagement Activity 2022
URL https://ispp2022.com/
 
Description Invited Talk (6th Early Career Research Symposium on Cyanobacteria, online ) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a research presentation online. ~70 people attended. Opportunity to disseminate research and advertise CyanoGate and CyanoSource.
Year(s) Of Engagement Activity 2021
URL https://eventform.plantae.org/plantae.calendar/detail/831/1636963200000
 
Description Invited Talk (CyanoWorld Seminar, online) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave a research presentation online. ~70 people attended. Opportunity to disseminate research and advertise CyanoGate and CyanoSource.
Year(s) Of Engagement Activity 2022
URL https://www.youtube.com/watch?v=ym_a5VqySVc
 
Description Invited Talk (Jagiellonian University 50th Jubilee Conference, Poland) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave an invited research presentation at Jagiellonian University. The conference was attended by >100 people, there was opportunities to disseminate research and advertise CyanoGate and CyanoSource, and explore collaborative ideas.
Year(s) Of Engagement Activity 2021
URL https://jubileuszwbbib.confer.uj.edu.pl/en_GB/krakow-i-uj
 
Description Invited presentation - Cyano 2023, University of Kassel, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Gave an invited research presentation at Jagiellonian University. The conference was attended by 50-100 ERCs and PIs, there was opportunities to disseminate research and advertise CyanoGate and CyanoSource, and explore collaborative ideas.
Year(s) Of Engagement Activity 2023
URL https://www.uni-kassel.de/tagung-konferenz/cyano2023
 
Description Invited talk at the Annual Conference of the French Photosynthesis Society, Paris, 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Gave an invited research presentation at the Annual Conference of the French Photosynthesis Society, Paris, 2023. The conference was attended by 50-100 people, there was opportunities to disseminate research and advertise CyanoGate and CyanoSource, and explore collaborative ideas.
Year(s) Of Engagement Activity 2023
URL https://sfphi2023.sciencesconf.org/
 
Description New species, tools and resources for progressing cyanobacterial biotechnology. Algae-UK 1st Annual Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Algae UK is a network supporting researchers and others interested in the exploitation of algal products and processes in industrial biotechnology.
Year(s) Of Engagement Activity 2019
 
Description Technical Webinar Phycocyanin from Algae 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact European Algae Biomass Association (EABA) webinar to bring together academics and industry working with phycobilisome products
Year(s) Of Engagement Activity 2020
URL https://algaeworkshops.org/phycocyanin-from-algae/