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Revision as of 01:37, 22 October 2019

I M P R O V E

Every Synbio Experiment is more or less based on the same principle: You change a system in some way and you look at the outcome. This readout is one of the most important things in all natural science, a wrong readout can easily flaw your whole experiment or can lead to serious misconclusion.
The most common way to measure localisation, interaction or even the intensity of genetic elements is via Fluorescence as readout.
Fluorescence Proteins (FP), started with the green fluorescent protein, are based on the ability of a chromophore to absorb photons of specific wavelength and emit this photon at another. Even on the iGEM registry, the characterization via FPs is the suggested way to characterise a part.
This Method is prone to Background noise, depends on the folding of the Protein at the specific cell conditions and furthermore the chromophore can even bleach after to much exposure, so the drawbacks are obvious.

Bioluminescence could make the desired difference, but the original Luciferase Assays either consistent of an whole Operon systems, or put an unnecessary high metabolic burden through ATP dependency and/or trough its relatively large size (Firefly-Luciferase 61,5 kDa). Together with the low quantity, which can be several orders of magnitude lower than a fluorescence based system, the common breakthrough of Lumincese in Synthetic biology is still missing.
Newly developed small ATP independent Lucferase Proteins, are interesting candidates to bypass these Problems. Nanoluc, with its 19 kDa and up to 150 fold increase in brightness compared to the Firefly-Luciferase is handled as an suitable alternative. This Protein use the patented Substrate Furimazine, and emits Photons at 460 nm. Naoluc has been successfully implemented in Promoter testing and as an alternative in Interaction messurement via Bilumiecnce Resonace energy transfer, but sadly only few team ever used this system.
One scratch on the surface of Nanoluc is for sure the restriction of the wavelength. While for Measurements in many organisms and Tissues, this looming Problem did not occur, it's becoming obvious, when looking into phototrophic Organisms and deep-tissue mammalian cells. As the keen reader might guess, cells absorb Light of the wavelength under 600 nm to a great extent and even more if they have a photosystem. Chlorophyll a have one their two peaks at 440 nm [fig.1]. If one would compare that with the nanoluc spectra, a devastating conclusion could be made: The Photosystem will absorb photons from the Signal, leading to weaker peaks, and maybe more grave/frightening/alarming, a dependency of Signal on the chlorophyll content. Althroug localisation experiments should´t be affected that much, Measurement and characterisation, the foundation of which synthetic Biology is build on, could be shaken. Driven by this problem, we dig ourselves in literature and found a solution: A mutated Version of nanoLuc, so called teLuc
BBa_BBa K3228042 BBa K3228042
which has a severe red shifted pattern with a peak at 502 nm (Figure 2). What is even more serviere is the astonishing brightness, wich even surpass nanoluc by several folds (5,7) in vitro. In vivo this effect is even more dramatic, through its ability to bypass the absorption of Light. We expect this ability of teLuc to surpass the limits of Luminescence in plants to an amazing extent, and allow the plant synthetic biology community to accelerate their research.

Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra
Fig.2 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra

Lvl1 ori
Fig.1 - Lvl1 ori

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.