Great things are not done by impulse, but by a series of small things brought together. - Vincent van Gogh

This year we expanded the Marburg Collection from 2018 with 55 new parts to the Marburg Collection 2.0. With our developed workflow we could characterize our parts and compare them with a second measurement method: FACS/flow cytometry.
We added two new features for genetic engineering of cyanobacteria: A CRISPR/cpf1 guided knockout system as well as a modularized assembly of repair templates for the knock in of genes (M.E.G.A. expansion).
This includes integration sites that target conventional neutral sites in cyanobacteria but we also rationally designed two novel integration sites based on RNA-seq data. Additionally, we offer the first MoClo compatible shuttle vector for cyanobacteria and characterized gene expression based on that origin of replication.
We used our new shuttle vector to build standardized devices for the characterization of BioBricks in cyanobacterial chassis to improve the reproducibility of results and to simplify large scale assemblies. For this we used placeholders, a novel part type that aids in the construction of a larger set of parts by reducing the involved cost and workload significantly. Additionally, we tested our toolbox with PCC 7942 to show that the Marburg Collection 2.0 is also working with similar cyanobacteria. We offer free access to the data of our characterization, enabling the iGEM community and scientists to choose the parts based on this data.
To improve the measurement method applicable for cyanobacteria we focused on measurements via luminescence reporters over fluorescence reporters, because of the fact that cyanobacteria emit autofluorescence.
This way our results are way more accurate, because of the reduced background noise. The higher accuracy is obviously visible during the measurement of our parts, where we could see a difference of 5x105 between the background noise and the signal, which implements that already a small amount of sample has a more intensive signal.
Hereby, we want to encourage the community of young scientists to work with the fastest phototrophic organism Synechococcus elongatus UTEX 2973 because of its high relevance for biotechnological applications.

Validated part

We successfully built and validated our new placeholder parts . These parts have a vital function in large scale Golden Gate assemblies. In some applications, like rapid prototyping, large scale characterization studies and screening methods of the assembly of Golden Gate constructs that only differ are one position is in big demand.
Placeholder cut down on the workload and the invested resources required for a large scale Golden Gate assembly. We ourselves validated their use and calculated the amount of work required to assemble a promoter library with 20 parts compared to the hypothetical workload when not using our placeholders. We validated the part by using it in our large scale assemblies. The use is clearly documented in our labfolder protocols (for more see: BBa_K3228073).

Evaluation of NanoLuc as a reporter

In conventional chassis such as E. coli or S.cerevisiae fluorescent proteins are often used as reporter genes to validate genetically modified organisms. However measuring fluorescent activity may not be the best option to choose for phototrophic organisms, because of the absorption caused by the absorption of the photosynthetic pigments of the host organism. The table below shows the absorption spectra of UTEX 2973. As one can see, reporters such as mTurquoise and YFP underlie a rather high background noise caused by the absorption from the pigments at their ideal wavelength (YFP: 515nm and mTurquoise: 445nm).

Fig.1: Fluorescent reporters mapped on the absorption spectrum of UTEX 2973.

NanoLuc promises to be a more ideal reporter gene, because there is almost no background noise for this measurement method. To characterize the chosen part from the registry we firstly measured the activity in E.coli before we went any further. We made a dilution series of the E. coli (DH5α) strains to determine the proper dilution for the measurement. After that we mixed 50 µl of culture with 50 µl of NanoLuc substrate.
Our results were remarkably good, because we needed to dilute the cultures 1:100 in order to get a signal that is under the maximum value, which our plate reader could handle. The results seen below were achieved with cultures grown to OD ~0,8 (600 nm) and the measure points were taken 39 minutes after treating the cultures with the NanoLuc substrate (see data here).

Fig.2: NanoLuc measurement with four biological replicates in E.coli DH5α