Team:Marburg/Basic Part
B A S I C P A R T S
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 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 we 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 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 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 46,4 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.
This small plasmid has already been used for construction of shuttle vectors (Kuhlemeier & van Arkel, 1987 ; Golden & Sherman, 1983 ; Chen 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
et al., 2016) and E.coli (Gerhart
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.
The results can be found here.