Synechococcus elongatus UTEX 2973

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Strain Engineering

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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.

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/Cpf1. 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.

Marburg Collection 2.0

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