The origin.

Inspired by the fast progress in synthetic biology and its urgent need for genetic tools that enable the exploitation of cyanobacteria for research and biotechnological applications, we set out to construct the most versatile shuttle vector for cyanobacteria based on the modular Golden Gate Assembly method, allowing for flexible cloning into a reliable self-replicating system.

Introduction of exogenous DNA can be done in multiple ways and propagated in a strain if it is integrated in the chromosome or stably expressed on a self-replicating plasmid.
For rapid prototyping in cyanobacteria self-replicating plasmids are of higher interest than genome-integrations, as the latter can be quite time-consuming in cyanobacterial strains with multiple genome copies (Griese et al., 2011). Furthermore, genes introduced in self-replicating vectors have been shown to have higher gene-expression levels than those integrated in the genome, as copy numbers are typically higher (Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016) – a desirable trait, not just for rapid prototyping in research applications, but also for biotechnological production of valuable compounds.
With our shuttle-vectors encompass a cyanobacterial origin of replication (ori) from Synechococcus elongatus PCC7942 as well as an E.coli ori, which is perfect for fast cloning processes, as these vectors can be easily recovered from the cyanobacteria and reintroduced in an E.coli strain.

Currently existing shuttle vectors for cyanobacteria are still based on standard systems working with multiple cloning sites (MCS) for expression of homologous genes (Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016). A huge downside is that these vectors include either an MCS (e.g. pAM5188) or a fluorescence reporter (e.g. pAM4787), which is unpractical for easy selection of recombinant clones. Additionally, an MCS comes with possible sequence constraints due to restriction sites leaving unwanted base pairs in your constructs.
Facilitating and standardizing the process of engineering biological systems is one of the fundamental goals of synthetic biology (Shetty Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2008), so the construction of a shuttle-vector based on a modular cloning method significantly improves the genetic toolbox we created for genetic engineering and synthetic biology approaches in S.elongatus and other cyanobacteria.

The commonly used S.elongatus strain PCC7942 carries two endogenous plasmids, the 46,4kb pANL (Chen et al., 2008) which is essential and the 7,8kb pANS (Van der Plas et al., 1992) which is not essential for the strain and can easily be cured.
This small plasmid has already been used for construction of shuttle vectors (Kuhlemeier Titel anhand dieser DOI in Citavi-Projekt übernehmen & van Arkel, 1987 ; Golden & Sherman, 1983 ; Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016).
We followed this lead to create the best shuttle-vector available for cyanobacteria by encompassing the minimal replication region of pANS and the ColE1 origin of replication into our vectors, allowing for stable self-replication with high copy numbers in cyanobacteria (Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen et al., 2016) and E.coli (Gerhart Titel anhand dieser DOI in Citavi-Projekt übernehmen et al.,2002). This addition to the genetic toolbox proves invaluable, as it can be easily recovered from the cyanobacterial strain and reintroduced in E.coli for fast GoldenGate-based cloning processes.

In order to supply the community with an easy selection system, we equipped our shuttle vector with a fluorescent reporter that is cut out when introducing new genetic parts:
A mRFP (red fluorescent protein) cassette is flanked by our standardized TypeIIS restriction enzyme recognition sequences (BsmBI or BsaI depending on what level you want to clone in). In a standard Golden Gate reaction this cassette will drop out and leave space for the parts that should be introduced, allowing for easy selection on plate after successful cloning – red colonies are wrong, still harboring the mRFP cassette and white colonies (if no other fluorescence is introduced) are correct, as the mRFP was switched with the parts of interest.

This crucial part comes in two variations - one for cloning Lvl1 and one for Lvl2 constructs -, giving the Golden Gate community everything they need for successful and reliable creation of self-replicating vectors in cyanobacteria.