Cloning design - 4C_FUELS

Nowadays the ever expanding toolbox of synthetic biology is providing us with new tools for easier genetic editing and novel strains with appealing features year by year. Intensive efforts are being performed to expand our ability to engineer phototrophic organisms. Interest in phototrophic organisms is growing, and it would not be disproportionate to say that we are on the doorstep of an authentic phototrophic revolution in the field of synthetic biology.

Within this page you will read about the phototrophs potential for biotechnological applications and how newly discovered strains could fuel the transition towards the large-scale utilization of phototrophic organisms aided by the means of synthetic biology.

Why using Phototrophs?

Phototrophic organisms are the beginning of life as we know it today. Despite their importance in the biosphere for inorganic carbon fixation, their utilization within synthetic biology has been limited. However, times are changing fast. Their attractiveness for carbon fixation purposes in the era of climate change, combined with novel discoveries are fueling a complete revolution within phototrophs utilization in biotechnology.

Despite the edition of phototrophic organisms genes is not new, genome alteration and phototrophs synthetic biology has lagged behind the biotechnological flagships: heterotrophic organisms. Nowadays it can be considered that model organisms such as Bacillus subtilis, Escherichia Coli or Saccharomyces genre have been extensively studied and engineered to serve for human purposes. On the other hand, the utilization of phototrophic organisms is still limited. The main reason is the lack of available edition toolboxes, standardized parts and information about its metabolism and cellular regulation.

However, phototrophic organisms are gaining momentum year by year. Its unique ability to synthesize products directly from carbon dioxide, light and water is an attractive feature that can help to simplify biotechnological production processes. SInce the amount of feedstocks required for the cultivation of phototrophs is reduced, the sustainability and overall efficiency of the phototrophic synthesis process can greatly surpass the one found in heterotrophic organisms.

Phototrophic production systems involve a smaller number of steps from the initial materials to the final products. This way, carbon and energy losses in each stage of the manufacturing chain can be reduced.


Because of this, phototrophs could be the game changers of the current biotechnological paradigm, leaving heterotrophic chassises for specialized applications or the recycling of organic waste. From the mass-production of safe and easy to store pharmaceuticals in genetically engineered plants, to the biomanufacturing of biofuels and chemical products at large scale relying solely on light and water. We are in the age where we are starting the keys to harness the real potential of phototrophic organisms. However, to achieve this titanic task, several challenges still need to be addressed.

While plants and superior organisms have found a clear niche for the cheap and easy production of high added value compounds, phototrophic microorganisms such as microalgae and cyanobacteria are seen either as a fine chemicals production platform to mere biomass to utilize as an organic material to process.

In any of these cases, it is clear that for the implementation of these organisms within our economic model, we still require the development of powerful edition toolboxes, collections of well characterized genetic elements as well as the domestication and standardization of robust strains for its industrial utilization.

The need of new chassis: PCC11801

In the case of phototrophic microorganisms, there is a big variety of laboratory strains, where each one of them is used because of its specific features. Even considering only cyanobacteria, differences between freshwater and marine or those strains capable or not of nitrogen fixation are big enough to offer a wide range of possibilities depending on the requirements of a certain application. However, in spite of this wide repertory of strains, their biological characterization and standardization is far away from other conventional chassis organisms, which eventually slows down the development of research in the field.

They promises of new phototrophic chassis

Likewise, the majority of these laboratory strains still lack key features required for its industrial utilization. The growing times are slow, cultures can be easily contaminated and many of them are not robust to withstand harsh conditions. Most of these strains have actually evolved within laboratories, selecting those traits that made them desirable for laboratory research, but sometimes comprising its ability to thrive in the environment.

But things are changing fast. The growing interest in phototrophic microorganisms is leading to the discovery of many novel strains with highly interesting properties, either for research and industrial purposes. Among them, the fast growing phenotype is a highly desirable train.

New Laboratory Strains

The combination of fast-growing phenotypes with natural transformation capacity is the ideal foundation for an easy manipulable laboratory strain.

Among cyanobacteria has been observed how fast-growing phenotype is usually conferred by a set of small mutations in the genome, capable of strongly altered the regulation and metabolic performance of the organism. In general terms fast-growing strains alter its utilization of cell machinery, routing most of the fixed carbon to biomass formation instead of storage compounds, even under light or carbon saturation conditions.

This is a crucial aspect, since it implies that all the already available knowledge in terms of static biological characterization of laboratory strains, could be combined with new discoveries found in novel strains in order to adapt or develop new organisms for research or end-application needs.

Novel chassis for industrial application

In the industrial application case, the set of desirable traits expands, considering: the ability to grow in a wide range of conditions, high tolerance to environmental stresses (high light, variations in temperature, presence of xenobiotics or pollutants, high salt concentrations, intermittent irradiation etc…) , and a fast-growing phenotype. In addition, sharing similarities with well-studied laboratoratory strains is also a crucial aspect that can greatly enhance the rapid adaptation of already existing protocols and tools to engineer these novel strains. Either for biomanufacturing or other applications such as biosensing, all of these traits will be required for more robust phototrophic biological engineered systems.

The different metabolism regulation found in fast-growing strains has been shown to improve the expression levels of endogenous and heterologous enzymes, as well as higher titers of in engineered metabolic pathways. Then even when an engineered pathway has been designed for a conventional laboratory strain, its adaptation to a fast-growing cyanobacteria could in fact improve its performance.

However, within the industrial context, the type of CO2 sources, light intensity in the area or the availability of fresh or seawater will determine the ideal organism to use. This way, having for each set of conditions, an ideal phototrophic chassis for industrial applications is a requirement.

Synechococcus elongatus PCC11801. An ideal chassis for freshwater cyanobacteria strains.

Among all of these emerging strains, Synechococcus elongatus is one of the most common genera to which these candidates belong. In particular, we have decided to utilize the fast-growing Synechococcus PCC11801. This strain gathers all the desirable requirements to become an useful phototrophic chassis for biomanufacturing as well as an ideal model chassis for freshwater cyanobacteria.

PCC11801 is a robust fast growing cyanobacteria that not only is capable of growing from 30 to 39 ºC and at atmospheric CO2 concentrations. It is also a strain which shares more then 80% identity with the widely studied Synechococcus PCC7942. What’s more another highly similar strain, Synechococcus PCC11802, shares more than 99% of its genome but it has demonstrated optimal growth under high CO2 concentrations. Because of that, this strain could become a robust model for the expression of engineered pathways.

The industrial perspective

Its highly active metabolism can be exploited for the expression of already designed pathways in related cyanobacteria with a higher product yield, as has been demonstrated in available literature. In addition, its tolerance to multiple pollutants and ability to withstand high temperatures and light intensities up to 1000 µE without experiencing detrimental effects makes this strain a really promising alternative for the industrial utilization in outdoor installations operated in warm climates.

A great choice for research


In addition, due to its high similarity (83%) with the well-known PCC7942, its natural transformation capacity and the ability to grow fast even at atmospheric CO2 concentrations makes this strain almost the ideal chassis for research and development for metabolic engineering. What is more, pathways engineered for PCC11801 could be easily transferred to PCC11802 and other related model strains such as the marine Synechococcus elongatus PCC7002 and PCC11901.

Due to all these characteristics, this strain can be easily manipulated, taking advantage of almost all existing edition tools. In addition, its robustness and rapid growth make it an ideal candidate for prototyping genetic constructs, even in laboratories with limited resources.

Finally, the similarity in its metabolic network and codon usage with other organisms with different phenotypes, make it a good test platform, where the pathways can later be exported to other organisms with a more relevant phenotype for the final application (greater CO2 capture or even growth in marine water).

Establishing PCC11801 as a chassis

Amazed by all the exciting features this novel strain has, we decided to design a toolbox that aims to ease the genetic manipulation of these and other related strains. To do so, we embarked on the adventure of accessing the strain which is currently protected by the Indian government under the Nagoya Protocol. After a really long process, we eventually managed to access the strain and committed to contribute to the community with the following projects.

First, we wanted to test if available replicative plasmids that have proven to work in closely related Synechococcus elongatus strains could also work in PCC11801. Likewise, we have optimized the transformation and cultivation procedures of the strain.

pANS origin of replication

pANS is a recently used origin of replication for cyanobacteria. It has been proved to work in freshwater Synechococcus elongatus strains, as well as in Anabaena 7120 and related organisms. We wanted to test if it will still work in PCC11801.

During the initial research on this strain, we discovered that the non-essential small endogenous plasmid from PCC7942 (pANS) had multiple sequences which shares a high identity degree with PCC1801 genome. After discovering how important genes are conserved between both strains, we thought that a recombination event of pANs plasmid may have taken place during the evolution of PCC11801. Then, the replication machinery for pANs ori could still be preserved within the genome or probably disappeared by the accumulation of mutations.

Our results after pANs transformaton demonstrated the appeareance of colonies when plated in low concentrations of antibiotic, however cells in these colonies were unable to grow well in fresh BG11-Agar plates or even proliferate in the nitrocellulose membrane filters used for transformation.

Second, we have developed a software for the identification of novel neutral integrations sites. Using this program, we have identified 5 potential neutral sites that could be used for genomic integration without affecting the fitness of the strain. (You can read more about it in our Software section.

Eventually, as a requirement for the large-scale utilization of engineered cyanobacteria, we have designed a strategy for the easy generation of markerless mutants via integrative genome edition. This tool aims to provide a fast, easy and widely compatible system for cyanobacteria markerless mutant generation, overcoming the limitations of producing strains which harbor resistance cassettes. You can read more about this in the Engineering section.

However, in spite of us starting all the tramits in March 2021, due to the long procedures required to access the strain, we were not able to access it till the beginning of september. With this limited time span and the issues we found during our cloning, we were not able to test all the developed strategies. Instead, we have proposed a solid theoretical design, and committed ourselves to promote the utilization of synechococcus PCC11801 as a robust model organism for cyanobacterial research.

Building a phototrophs research community


Since the very beginning of this iGEM season, we wanted to promote the utilization of phototrophs. We were surprised that, despite growing, phototrophic synthetic biology was still underrepresented in previous iGEM seasons. Then we started looking for other teams which shared our interest, in order to create a network of phototrophic-based iGEM projects, and establish the foundations of an authentic community of phototrophs within iGEM. In the beginning we met with other phototrophic-related teams such as LMSU ,IISER_Pune or MiamiU_OH, looking for ways to collaborate together and create a stronger phototrophic community.

During spring of 2021, we discovered that Marburg and Bielefeld-CeBiTec iGEM teams were already working together for the same purpose. After receiving an invitation from their side and discovering the impressive work they have already made, we decided to join their project and start working together.

This ambitious collaboration of 11 different teams, ended in the creation of a strong phototrop community within iGEM. During 2021 the community has been structured among three main pillars. First, slack workspace for networking and keeping active communication. Second, a meetup series, where experts in phototrophic synthetic biology and teams shared their experience, knowledge and current opinions about the present and future of this exciting field. Eventually, decided to make this initiative the beginning of a solid reality across the different iGEM editions, a handbook has been created. This handbook summarizes this year’s experiences to the future generations of phototrophic iGEM teams, providing them with useful information, tools and protocols for their future projects.

Excited by this collaborative project, we contributed to the strengthening of iGEM Phototrophs community by taking part in all the meetups, sharing our project ideas with other teams and writing in the handbook.


Excited by this collaborative project, we contributed to the strengthening of iGEM Phototrophs community by taking part in all the meetups, sharing our project ideas with other teams and writing in the handbook.

You can download the first edition of the handbook here:

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Sengupta, S., Jaiswal, D., Sengupta, A., Shah, S., Gadagkar, S., & Wangikar, P. P. (2020). Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid. Biotechnology for Biofuels 2020 13:1, 13(1), 1–18.

Jaiswal, D., Sengupta, A., Sohoni, S., Sengupta, S., Phadnavis, A. G., Pakrasi, H. B., Wangikar, P. P. (2018). Genome Features and Biochemical Characteristics of a Robust, Fast Growing and Naturally Transformable Cyanobacterium Synechococcus elongatus PCC 11801 Isolated from India. Scientific Reports, 8(1).

Jaiswal, D., Sengupta, A., Sengupta, S., Madhu, S., Pakrasi, H. B., & Wangikar, P. P. (2020). A Novel Cyanobacterium Synechococcus elongatus PCC 11802 has Distinct Genomic and Metabolomic Characteristics Compared to its Neighbor PCC 11801. Scientific Reports 2020 10:1, 10(1), 1–15.

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