Synechococcus elongatus UTEX 2973: A review
Introduction
Cyanobacteria have been popular in research for centuries but recently they gained a spotlight in Synthetic Biology. The forefather of photosynthesis is interesting because of its simplicity, making it easier to engineer the system but also because of its growth speed that surpasses that of plants. In recent years phototrophs became the notorious revolutionizers of “green biotechnology”: as photoautotrophic organisms, they only require CO₂ and sunlight as carbon and energy sources to generate biomass.

The following introduction serves as an overview over our new chassis Synechococcus elongatus UTEX 2973, based on the latest research results.

The organism
The gram-negative photoautotrophic cyanobacterial strain Synechococcus elongatus UTEX 2973 is an isolate from the 1955 described strain Anacystis nidulans. This strain was kept at the University of Texas as Synechococcus leopoliensis UTEX 625. A colony was selected from a mixed culture of this strain, resulting in Synechococcus elongatus UTEX 2973. The resulting organism is genetically very close to the well studied strain Synechococcus elongatus PCC 7942. With the fastest measured doubling time of below 90 minutes and a high tolerance to temperature and light intensity, UTEX 2973 is a chassis to keep an eye on. Cyanobacteria have big advantages compared to other phototrophic organisms such as plants or eukaryotic algae: next to their faster growth they also convert solar energy a lot more efficiently. The faster generation of biomass makes cyanobacteria a potential candidate for biotechnological application and their amenability to genetic modifications (Ungerer, Wendt, Hendry, Maranas & Pakrasi, 2018) make them a great platform for research. Despite these advantages, cyanobacteria have still not arrived in Synthetic Biology quite as we want. With our highly optimized chassis Synechococcus e. UTEX 2973 we want to change just that.

Comparison to the well-studied organism Synechococcus elongatus PCC 7942

Genome sequencing has proven that our strain is 99.8% identical to the much better studied strain Synechococcus elongatus PCC 7942, which is surprising since both strains were isolated from completely different locations. While UTEX 2973 tolerates high light intensities, PCC 7942 is photoinhibited by light intensities of less than half of those which UTEX 2973 can withstand. In electron microscopic examinations carboxysomes and polyphosphate bodies were found in both strains. Most conspicuous are the spherical, 30 nm sized electron-dense bodies in PCC 7942, which are not present in UTEX 2973. It is assumed that the bodies are carbon stored in the form of glycogen. UTEX 2973 does not generate glycogen storage and uses the carbon directly for biomass production, resulting in faster growth.
Research has also proven that several changes in the photosynthetic apparatus cause decreased phycobilisomes but enhancement of Photosystem I, cytochrome f and plastocyanin contents (Ungerer et al., 2018) .
The most notable advantage is UTEX’ unparalleled doubling time. PCC 7942 takes more than twice as long, while only producing a third of its biomass. In an experiment under the same initial conditions, the dry weight of UTEX 2973 also increased to 0.87 mg/ml, compared to only 0.33 mg/ml in PCC 7942 (Yu et al., 2015) . Unlike UTEX 2973, PCC 7942 is naturally competent due to its porin-like proteins. These proteins are encoded on the inverted region in the genome so that inversion in UTEX 2973 can be reversed.

But what allows UTEX 2973 to have such vital advantages?

When comparing both strains, one can observe that their content of amino acids varies greatly: the amount of amino acids in UTEX 2973 lies at 53% whereas in PCC 7942 it is 40.9% (Mueller, Ungerer, Pakrasi & Maranas, 2017) . This results in a different composition of the biomass, which is due to the discovered single nucleotide polymorphisms (SNP’s). Among other things, they cause an increased translation rate through a more efficient RNA polymerase. In addition, UTEX 2973 increases.

Molecular aspect
UTEX 2973 differs from PCC 7942 in 55 single nucleotide polymorphisms and insertion-deletions, as well as a 188.6 kb inversion and a six open reading frame deletion (Mueller, Ungerer, Pakrasi & Maranas, 2017) . Thereby these mutations must contain the genetic determinant for UTEX’ rapid growth rate.
Three genes have been discovered as potentially being involved in better growth. AtpA encodes for the alpha subunit of an ATP-synthase with an apparent higher specific activity. The difference results in a substitution of one amino acid (Cys in PCC 7942 to Tyr in UTEX 2973). Another significant difference lies in the ppnK encoded NAD+-kinase. Glutamin acid in PCC 7942 substitutes into aspartic acid in UTEX 2973, which affects improved enzyme kinetics. Another important gene is rpaA, which improves the circadian response regulator. These adjustments relieve a photosynthetic bottleneck, increasing the capacity for photosynthetic electron flow. This ensures the usage of higher light intensities while producing more ATP and NADPH to fix CO₂. All this ultimately leads to better growth of UTEX 2973. In this experiment, the SNP’s in UTEX 2973 were inserted into PCC 7942, thereby significantly improving its growth rate.
Thus, these minimal genetic changes cause a huge impact in growth rate and CO₂ absorption, which proves UTEX 2973 to be second to nothing, photorespiration and the synthesis of glyoxylate, a precursor of several amino acids. Some SNPs alter kinetic parameters of metabolic enzymes and increase the production of biomass components. Most striking is the difference in the rate of carbon uptake and its allocation in biomass. UTEX 2973 absorbs CO₂ 2.06 times more efficiently than PCC 7942.

Field of application
Synechococcus elongatus UTEX 2973 has the potential to change paradigms in Synthetic Biology:
With its incredibly low doubling time for phototroph standards, UTEX 2973 is eligible for rapid prototyping.
We need good innovations to accelerate feasible photosynthetic research. Phototrophic organisms such as plants are simply too slow inhibiting innovative progress. With our organism UTEX 2973 it is possible to test different parts and plasmids quickly, inexpensively and easily.
Cloning is a time-consuming process, especially when you have hundreds of parts in a collection. You can quickly test all the parts in UTEX 2973 and then test the working assemblies in your actual organism, so UTEX acts as a technical prototype to accelerate the design build test cycle.

If an assembly didn't work, it's easy to locate the error, optimize the construct design and get the desired result quickly.
As a photoautotrophic prokaryote, UTEX 2973 is a simple organism that is easy to work with. Plants as eukaryotes are much more complicated, so the work and research become more complex.
So if you want to advance photosynthetic research, you can, for example, analyze and modify pathways on this prokaryot without much effort. UTEX 2973 is perfect to design engineering and principles in an easy chassis and afterwards apply it all to a higher organism.
UTEX 2973 can also be used to significantly enhance the process of characterization and standardization of cyanobacteria and their biological “parts-list”.
We have already advanced this aspect and established a reproducible “parts-list” so it is already possible to easily work with it.

In "Green biotechnology” our chassis can be used to sustainably produce carbon neutral platform chemicals without fossil fuels. These platform chemicals can be used to produce biofuels and bioplastics or carbohydrate feedstocks (Song, Tan, Liang, & Lu, 2016) . Innovations like these are needed to propel the development of a sustainable, fossil fuel independent industry of tomorrow. Many secondary metabolites have pharmaceutical benefits, such as amino acids, fatty acids, macrolides, lipopeptides and amides (Yu et al., 2015). The cyanobacteria strain Synechocystis sp. PCC 6803 already serves as a brilliant example for the application of cyanobacteria: it has been genetically modified to secrete fatty acids and thus to avoid costly biomass recovery in the production of photosynthetically produced, sustainable biofuels (Liu, Sheng, & Curtiss, 2011) .

Establishing UTEX 2973 with its versatile capabilities could pave the way to making industrial biotechnology more sustainable and thus be a solution to combating climate change, one of the most horrific challenges humanity has ever faced. UTEX 2973 embodies progress and innovation on the highest level.

Genetic amenability
In recent years, the CRISPR system has enabled precise gene editing. Gene editing is well feasible in UTEX 2973 with the CRISPR technology and an alternative nuclease Cas12a from the organism Francisella novicida as Cas9 is toxic to cyanobacteria (Ungerer & Pakrasi, 2016).
Despite the loss of its natural competence, UTEX 2973 is a suitable candidate for genetic engineering. DNA can be easily introduced into UTEX 2973 trough triparental conjugation via E. coli and the self-replicating vector pANS. Shuttle vectors are of great interest as they lead to higher gene expression compared to genome integration. In addition, they retain large DNA inserts in the organism even without selection pressure and are easy to transform (Chen et al., 2016). We developed facile-transforming shuttle-vectors and thus, we were able to extend the genetic toolbox and simplify genetic engineering.

Natural Competence

One of the most important aspects when engineering an organism is the actual modification of its genetic code. The introduction of exogenous DNA can be done in multiple ways - through electroporation, conjugation, heat shock or via natural competence.
Electroporation is a method in which an electrical field is applied to cells, in order to increase the permeability of the membrane, enabling DNA uptake not just in prokaryotic (Thiel and Poo, 1989), but also eukaryotic cells (Potter and Heller, 2010). Although relatively simple to perform, this technique is not ideal, as the success rate is rather low and many precautions have to be taken: salt concentration and field strength highly effect the outcome and secreted endonucleases can degrade the DNA beforehand (Zeaiter et al., 2018).
Conjugation is a more complicated and laborious method where cell to cell contact is needed. Pili are formed to transfer DNA from one cell to another - but not all DNA can be transferred, as the plasmid that is to be conjugated needs to harbour a mobilization sequence (Actor, 2012). This method is more popular in cyanobacterial research, as it overcomes the above mentioned problems that come with electroporation (Zeaiter et al., 2018).

And what is natural competence?

Natural competence was first discovered by Frederick Griffith in 1928 (Griffith et al., 1928) by studying different Streptococcus pneumoniae strains - a virulent and a non-virulent one. Although he did not know the biological processes behind it, he realized that genetic information can be passed on from one bacterium to another, as previously non-virulent strains could be transformed to virulent ones. In comparison to other types of competence that can e.g. be chemically induced, natural competence is the ability of cells to take up extracellular DNA from their environment under natural conditions.

Natural competence is found in different kinds of bacteria - also in cyanobacteria. Despite the fact that there is still much to uncover about the exact mechanisms of natural DNA uptake in cyanobacteria, previous efforts by Schuergers and Wilde, 2015, Yoshihara et al., 2001, Bhaya et al., 2002 and many more have led to the construction of a preliminary model of the type IV-like pilus responsible for natural transformation in Synechocystis sp. PCC 6803 (Wendt and Pakrasi, 2019), which can be seen in Figure 1.

In order to take up extracellular DNA several steps seem to be necessary: The double stranded DNA has to be picked up by the type IV-like pilus and transported through an outer membrane pore comprised of PilQ subunits (Bhaya et al., 2002) and is then converted to single stranded DNA by a certain nuclease before being passed through an inner membrane pore composed of ComE subunits (Yoshihara et al., 2001).

Early studies have tried to identify cyanobacterial strains capable of natural transformation, from which just a few species have been frequently chosen to serve as model organisms, including Synechococcus sp. PCC 7002, Synechococcus sp. PCC 6803, Synechococcus elongatus PCC 7942 (Koksharova and Wolk, 2002).
Cyanobacterial species are known to be naturally competent, yet have been shown they have at least one complete set of the genes identified in the above mentioned model (Wendt and Pakrasi, 2019) - one of them being Synechococcus elongatus PCC 7942. This strain is closely related to Synechococcus elongatus UTEX 2973, in fact genome comparisons show just 55 single nucleotide polymorphisms and indels (Yu et al., 2015). Previous studies have suggested that a single point mutation in the pilN gene is responsible for the loss of natural competence in S. elongatus UTEX 2973 (Li et al., 2018), so in an effort to reintroduce natural competence into this strain, intact versions of the pilN gene from S.elongatus PCC 7942 have been successfully introduced into one of the neutral sites in the genome of UTEX 2973 via homologous recombination (Li et al., 2018), showing that natural competence can be achieved in this strain.

As natural competence is the easiest and often most efficient way to incorporate exogenous DNA into an organism, this is a crucial feature that comes in handy for every chassis.
For this reason we planned to restore the natural competence of S.elongatus UTEX 2973 - what approaches we took and how we planned all of it through can be found in our design section.

CRISPR gene editing

Clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR/Cas) systems are adaptive immune systems in bacteria and archaea that provide sequence-specific targeting of genetic sequences, in order to cut exogenous DNA (Barrangou et al., 2007).
Simplified systems have been a rising interest for use in genetic engineering approaches, as they can be used as powerful tools for precise genome alteration not just in prokaryotic, but also eukaryotic cells. This includes integration of whole genes, alteration of single nucleotides, knock-outs of whole genetic regions, as well as the use of the DNA-binding property in a multitude of applications through so called deadCas systems, where the Cas protein does not exhibit nuclease activity (Hsu et al., 2014).

These adaptive systems incorporate invading DNA sequences, so called protospacers, into their CRISPR array, meaning that short sequences of DNA can be stored between identical repeat sequences. This whole array is transcribed into a long precursor CRISPR RNA (pre-crRNA) that is then processed into mature crRNAs that carry spacers which serve as guides, leading the Cas protein to their recognition sequence, where it can then exhibit nuclease activity (Hille and Charpentier, 2016). Maturation of crRNAs differs in different CRISPR/Cas systems. CRISPR/Cas9 systems that are widely used in genetic engineering approaches need an additional transactivating crRNA (tracrRNA) for crRNA maturation, while in CRISPR/Cas12a (also called CRISPR/Cpf1) only the crRNA is necessary for precise targeting (Gao et al., 2016).
Another crucial factor of these systems are the protospacer adjacent motifs (PAM). In order for the Cas protein to effectively bind the targeted DNA sequence, it has to be next to a PAM sequence, proving that the PAM is an invaluable targeting component that allows the cell to distinguish between self and non-self DNA, as the PAM sequences cannot be found in the CRISPR array itself, preventing the Cas protein to cut inside of it (Mali et al., 2013).

As mentioned before, different CRISPR/Cas systems are available for genetic engineering of a large number of organisms. The most commonly used system is the CRISPR/Cas9 system, but another attractive system is CRISPR/Cas12a, also called CRISPR/Cas12a. The main differences are that Cas9 introduces blunt ends when cutting DNA, while Cas12a produces sticky ends and that Cas9 requires a tracrRNA for crRNA maturation, while Cas12a only needs the crRNA as a guide. In Cas9 systems the crRNA:tracrRNA duplex can be linked to form a single guided RNA (sgRNA), which is usually ~100nt long - in comparison the Cas12a crRNA is only ~43nt long (Swarts and Jinek, 2018).

Both systems are of particular interest for genetic toolboxes, as they enable highly accurate genome engineering with a wide application range - including multiplexed alterations.

Cyanobacterial shuttle vectors

Cyanobacteria are known to contain multiple copy numbers of their chromosome, the unicellular cyanobacteria Synechococcus elongatus reportedly contains 3-5 (Griese et al., 2011), 2-10 (Watanabe et al., 2015) or 1-10 (Chen et al., 2012) chromosomes per cell, more recent studies have counted eight chromosomes per cell (Yu et al., 2016).
S. elongatus PCC 7942 furthermore hosts two endogenous plasmids. The 46,4 kb pANL (Chen et al., 2008) which is essential and the 7,8 kb pANS (Van der Plas et al., 1992) which is not essential for the strain and can easily be cured (Lau & Doolittle, 1979).

In Synthetic Biology multiple approaches can be chosen to introduce exogenous DNA into an organism. Typically this is done by integrating the DNA into the host genome or by transforming plasmids that contain the genes of interest.
As we learned above, the copy number of the chromosomes can be highly variable, which is a huge downside when trying to engineer such organisms, as genome integrations have to be introduced into every single copy.
Shuttle vectors on the other hand can be stably maintained in the cells and are typically found in higher copy numbers, resulting in higher gene expression rates as genomic integrations (Chen et al., 2012).
Vectors that can be used in cyanobacteria are scarcely available, the few existing ones are mostly based on the RSF1010 plasmid that shows a broad host range and can be maintained in multiple cyanobacterial species, although for unknown reasons it is not present in high copy numbers (Mermet-Bouvier et al., 1993). The only shuttle vectors available that contain a native replication element of a cyanobacterial species are those that have been constructed from the previously mentioned pANS plasmid (Kuhlemeier & van Arkel, 1987; Golden & Sherman, 1983). Recent studies have shown that pANS-based shuttle vectors are present in a higher copy number than RSF1010- or pDU1-based vectors in cyanobacteria, clearly indicating the advantages of native replication elements. The same study has also proven that gene expression levels are higher when genes are expressed on the pANS-based vectors, than on pANL or the chromosome of S. elongatus (Chen et al., 2012).
In spite of these apparent advantages, many still prefer integrating DNA into the chromosome, which is why we incorporated parts for homologous recombination into our toolbox and successfully identified new neutral side for integration. providing invaluable tools for the community.

As we are certain that self replicating vectors are essential for many workflows, especially if rapid prototyping is to be done in an organism, we set out to construct the world's first MoClo compatible shuttle vector for cyanobacteria based on the modular Golden Gate Assembly method, allowing for flexible cloning into a reliable self-replicating system. With our constructs there is no need for tedious selection processes that come with genomic integrations.

Golden Gate Cloning and Modular Cloning: A historical review

Golden Gate assembly is a novel cloning method. It is at the heart of Synthetic Biology as it reflects the philosophy behind this area more than anything else. To really understand the mechanics and philosophy behind it, one has to look not only at the molecular basics but also at its history. This cloning strategy is based on Type IIS restriction enzymes. These enzymes have the uncommon property to cut next to their recognition sites, allowing the user to generate short DNA overhangs of their choice. This allows to seamlessly fuse DNA molecules together (Engler et al., 2008) . Another advantage is that the restriction sites can either remain or be completely cut off after restriction, based on the way the user decides to integrate a restriction site. This for example makes it possible to digest a fragment and ligate it in the same reaction without a chance that the fragment can be cut out again. This simultaneous restriction and ligation process is frequently termed „Golden Gate reaction” (Engler et al., 2009).

Pioneers in the field started to use these advancements to introduce a syntax into cloning procedures: while researchers were previously bound to use a variety of restriction enzymes, they can now break it down to two enzymes usually. By standardizing at which state of a cloning procedure which specific enzyme in conjunction with a specific entry vector is to be used, the process of cloning becomes more streamlined and researchers are given more time to focus on the vital questions of their endeavor rather than the particularities of cloning. The ability to produce overhangs of choice gave rise to the idea to standardize these overhangs based on the function of a genetic device. Early on, synthetic biologist saw how such a syntax complies with their philosophy of understanding genetic components as devices and soon they started standardizing overhangs for sequences like promoter, ribosomal binding sites and other part “types” (Weber et al., 2011). In this way, parts of different genes could be fused together effortlessly. It essentially allowed the cross compatibility of any genetic device in any organism, even across laboratories as international standards started to be become popular very soon. Singular devices like promoters were called “Parts” in analogy to machine components in engineering, further rooting the philosophy of Synthetic Biology in this cloning strategy. This type of modular assembly of parts via Golden Gate cloning is nowadays coined as “Modular Cloning” (Weber et al., 2011). Many part collections were published across the years, giving users full access to a big amount of parts characterized in their promoter strength, isolative capabilities and so on. Applicants were able to create vectors from scratch using DNA parts in conjunction with a complete data set on the activity of the parts to custom design the plasmids they need for their specific application. So many great thinkers advanced the progress in Modular Cloning and all of their works were vital to carry us to the point at which we stand these days. Here we present those that have influenced the design of our Part Collection, the Marburg Collection, the most.

Modular Cloning (MoClo) by Weber et al. (2011)

The modular cloning system was the first proposing a standard for Golden Gate based assembly. This toolbox offers five types of modules designed mainly for eukaryotes. The modules are stored in level 0 acceptor plasmids derived from the pUC19 backbone with a spectinomycin resistance and a LacZα cassette as dropout for blue/white screening. Custom level 0 plasmids are assembled by flanking the sequences with BpiI recognition sites and setting a single restriction-ligation-reaction with the correspondent plasmid. Up to five level 0 modules are assembled in an acceptor plasmid with ampicillin resistance and a LacZα-dropout by restriction with BsaI to transcription units. Six transcription units can be assembled using BpiI into level 2 multigene constructs containing a kanamycin resistance and a Cred-dropout. Alternatively Esp3I can be used to transfer the constructs to intermediary levels to reach higher levels for assembly of bigger constructs. This toolbox is best for constructs up to level 2 plasmids. Higher levels can be reached through intermediary levels but need two restriction enzymes for the assembly. Furthermore, it is important to know in which level the current constructs stand to avoid messing up the acceptor plasmids for subsequent levels. One of the (in our opinion) best executions of a Modular Cloning system is the yeast toolkit, also known as the Dueber toolbox (Lee et al. 2015). It offers a Golden Gate based system adapted for yeast. The basic level 0 parts are classified in eight types with optional subtypes. New basic parts are assembled into entry plasmids by restriction with BsmBI. For building level 1 cassettes at least eight parts are assembled by a restriction-ligation step using BsaI. The innovation of this toolkit compared to the previous is the use of connector sequences for level 1 and higher assembly steps. This way plasmids for yeast can be build de novo without the need of a defined backbone. Furthermore, they integrated a method for simple chromosomal integration by linearization of the plasmids with NotI. On top of this the connectors can be used as homology sequences e.g. for ligation-independent cloning, Gibson assembly, ligase cycling reaction or yeast in vivo assembly.

The PhytoBrick standard: The Syntax of Syntex

Another significant milestone is the PhytoBrick (Patron et al., 2015) standardization. It offers a wide standard compatible with popular systems like MoClo aiming to create a standard focused primary on plant engineering efforts. The iGEM competition already accepted it as an standard and offers support for building parts designed for plants, yeast and bacteria. This system proposes twelve defined fusion sites applicable for the different genetic modules. The fusion sites are divided into three major classes for promoter parts, transcribed regions and terminator parts. These classes are divided into subclasses giving the flexibility to use optional modules like tags, promoter, regulators and enhancer regions. The system also proposes two types of universal acceptor plasmids (UAPs) derived from the pSB1C3 plasmid where level 0 modules can be inserted by a single restriction-ligation step with BpiI or BsmBI respectively.

Marburg Collection 2.0: The green expansion

We expanded on the Marburg Collection, a toolbox established by iGEM Marburg in 2018. Thanks to its broad host range design inspired by the “Dueber toolbox” from Lee et al. 2015 we were able to apply it to our new chassis Synechococcus elongatus UTEX2973. The design is extremely simple: LVL 0 parts are the basic foundation, they contain one promoter, ribosomal binding site or terminator etc. Up to eight LVL 0 parts are used to create a LVL1 plasmid with a single transcription unit. The Marburg Collection 2.0 presents a set of new parts adding several new functions, expanding the range of hosts to use our parts on over the genera of cyanobacteria as well as supporting new design options such as Placeholder assemblies and vectors for genomic integrations.

Enabling high throughput assembly with flexible placeholder parts

Some applications require the construction of an array of higher LVL parts that only differ in one part. We ourselves encountered this when we screened the promoters of the Marburg Collection in our new chassis: These plasmids all were the same except for a different promoter. A “placeholder” is a part that gets assembled in a LVL1 construct just like any other part. Internal cutting sites however make it possible to cut this part out in a second cloning cycle in order to replace it with a non-placeholder part of the same type. The advantages of using placeholder in high throughput assemblies are clear: A seven part assembly usually requires to screen multiple colonies before you find the right one, meaning that a lot of test digesting or sequencing is involved. This is feasible if you want to construct only a few parts. For high throughput assemblies, however, the cost and time does not scale well enough. A two part assembly however has an extremely high success rate, meaning that in most cases it is sufficient to just pick one colony to get correct sequencing results.

This two step assembly heavily cuts down the invested workload and the cost per sample. We designed these placeholders so they could aid us in our assemblies. By removing limiting cost and time factors with a smart design option we managed to close a big bottle neck on the way to upscaling Modular cloning. Aside from a use in screening, these parts can also be utilized to find new sequences with a function: A set of mixed together defined oligonucleotides with randomized bases can be inserted into a test vector containing a placeholder. This library of test vectors is introduced into a host to test the biological characteristics of that sequence. A fluorescence reporter on the vector can be used to sort out cells with the intended characteristic, for example in an adequate high throughput screening method like FACS. This massively accelerates the search for parts with a desired quality. Such brute force approaches are becoming very popular in recent Synthetic Biology (Smanski et al., 2014).

A small part in our Collection, a big application for the future

Just until recently Synthetic Biology was lacking a genetic platform for cyanobacterial hosts: The introduction of the panS based self-replicating shuttle vector marks the first useable plasmid (Chen et al., 2016), however it is not MoClo compatible. Therefore BBa_K3228069 is in our eyes the most important addition to our Marburg Collection 2.0. This part contains the minimal replication region of panS for cyanobacteria and a spectinomycin cassette; additionally the ColE1 origin of replication can be used for cloning in E. coli and V. natriegens. A second version with different flanks and a kanamycin resistance enables the construction of LVL 2 plasmids that can contain up to seven genes. We utilized the broad host-range flexibility of the Marburg Collection to add a full set of organisms to its list of applicable hosts. These parts are the heart piece of the green expansion as they describe the world's first MoClo compatible shuttle vector for cyanobacteria.

Characterizing parts for our new chassis

To make sure that scientists are able to use our toolbox as convenient as they do now with Vibrio natriegens, it is necessary to characterize our part collection for our new chassis. We established a workflow suited to cyanobacteria to characterize all our parts in a consistent way. We realized that with a phototrophic chassis we needed to rethink some common procedures to respect species specific requirements. Before the actual measurements many pretests such as establishing growth conditions in well plates had to be done. We evaluated many possibilities regarding growth of precultures and measuring procedures and present you the best way to measure activities in UTEX 2973.

Modular Engineering of Genome Areas (M.E.G.A.)

While a plasmid based introduction of genes is the most common way to introduce genes into a species, genomic integrations are also a highly demanded application. Often genes develop a very different phenotype in genomic contexts due to a lower copy number and interactions with neighbouring regions. The knockout of a gene by inserting a sequence in its position is also a well approved way to study genetic interactions in an organism. Our M.E.G.A. expansion enables the user to design vectors that can insert one or more genes into an integration site on the target genome. Next to three conventional integration sites for cyanobacteria (NSI to NSIII) that are used worldwide (Holtman et al., 2005) we used a rational design approach to create two new ones (artificial neutral integration site options, aNSo I and aNSo II) that, according to RNA-sequencing data (See: design of integration sites in modelling), don’t show any transcriptional activity from neighboring genes. Therefore they are perfect candidates for a stable expression independent from cellular contexts.

Using the input from our bioinformatical analysis we can now provide the tools to engineer the genome of many cyanobacterial strains in a modulated fashion. Thanks to this expansion nothing stands in the way of tailoring custom strains to specific demands, be it of academical nature for Synthetic Biology and foundational research on photosynthesis or for industrial applications such as the design of producer strains for biotechnological processes.

Presenting a broad range arsenal of reporters for the green expansion

Reporters are an essential basic tool of Synthetic Biology. We present a set of reporters for a broad range of applications: From cyanobacteria specific well established reporters like sYFP to mTurqoise, an alternative than be used in conjunction with YFP for a dual fluorescent reporter system (best composite part) we offer a variety of fluorescence based reporters for part characterizations. To harness the incredible potential of novel findings in luminescence, we also provide a set of luminescence reporters based on NanoLuc, that strike out as completely cell independent, orthogonal reporters: The mutated version teLuc is especially well suited for cyanobacteria as it bypasses the natural absorption of cyanobacterial photopigments and Antares2 uses a FRET system that makes it possible to combine it with NanoLuc as a dual luminescence reporter system. Additionally we provide two reporters that have the ability to sense two very crucial cellular parameters in cyanobacteria: Phluorin2 for the detection of intracellular pH values that are crucial for rapid growth and rxYFP for the detection of the redox status, that can have crippling effects on cellular effects by damaging DNA, lipids and proteins through reactive oxygen species (ROS).

With rising atmospheric CO₂ concentrations and declining oil reserves, it is painfully obvious that the worldwide effort to change from petroleum-based industry to carbon neutral industry needs to accelerate drastically. One of the most promising key technologies right now is the use of phototrophic organisms for biotechnological applications. Hence, we decided quite early this year to devote ourselves to a project revolving around phototrophic organisms. During the design phase, we looked at different potential chassis like the model moss Physcomitrella patens , but soon stumbled upon many common obstacles that are characteristic for phototrophic chassis: time intensive culturing and complicated techniques to perform basic molecular biological methods. This showed us why every year, only very few iGEM teams decide to use a phototrophic chassis. As research on phototrophs is key to deeply understand and better engineer autotrophic organisms that offer powerful possibilities for a more sustainable future, we saw a need to tackle these issues. Inspired by the fundamental goal of Synthetic Biology to simplify the process of engineering biological systems we submerged into the world of cyanobacteria, soon realizing that one of the underlying aspects of engineering seemed to be missing: standardization. This process is vital to create reproducible results and achieve better compatibility and interoperability throughout the scientific community. Fueled by the discovery of this missing piece in cyanobacterial research we ventured out to establish Synechococcus elongatus UTEX 2973 as the fastest and most accessible phototrophic chassis to date, streamlining workflows wherever possible.

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