Do you want to explore the potential of cyanobacteria but don't know how to cultivate them?
We were in a similar place when Prof. Dr. Annegret Wilde (University of Freiburg) suggested giving the CellDEG cyanobacteria cultivation system a go. It turned out easy to use and our Synechococcus elongatus PCC 7942 grew happily.
Therefore, we want to help you at the start of the cyanobacteria cultivation journey by sharing the growth medium we used + how to assemble the CellDEG (HDC.10B) cultivators. Lastly, we want to thank Hess lab (University of Freiburg) for kindly sharing the FOM medium recipe and tips on how to use the CellDEG cultivation system.

You are looking for an easy-to-use, foolproof SDS-PAGE and Western Blotting protocol?
We have prepared it for you: a step-by-step guide with pictures and tips to make protein detection and analysis easier. With it, we hope to save you some of the bitter, time-consuming lessons we learned through using these methods several times during our iGEM journey.

Credits to the iGEM Freiburg 2022 team for teaching us How-To-Western-Blot and sharing their tips with us!

Our project involved testing cyanobacteria model strain Synechococcus elongatus PCC 7942 (referred to as PCC 7942) as a potential chassis for vitamin B12 production. PCC 7942 naturally produces pseudocobalamin but the insertion of only 2 genes, bluB and ssuE, should (in theory) enable endogenous production of the bioavailable B12 (as indicated by iGEM UCSC 2017 ; and further investigated by one team member, McKenna Hicks [1]). To modify PCC 7942, we decided to use a cyanobacteria-specific shuttle vector developed by iGEM Marburg 2019 (BBa_K3228069) which they kindly shared with us. This shuttle vector comes with 2 origins of replication (Ori): an Ori from PCC 7942, more specifically, from one of its endogenous pANS plasmids (which makes the vector compatible with our strain), and a high copy number Ori from E. coli, ColE1 (for more information, visit iGEM Marburg 2019 page.)

First, we cloned the aforementioned genes for B12 production (bluB and ssuE) into the shuttle vector, creating a new plasmid, piG_CBM. Next, we attempted to modify PCC 7942 with the piG_CBM via electroporation, conjugation (tri-parental mating), and natural transformation- none of which were successful (we observed no colonies on the plates containing antibiotic resistance). The reason for conjugation not succeeding was eventually found: the shuttle vector does not encompass an OriT (basis of mobility region/bom site) which would need to be added for the conjugation. Furthermore, according to Encinas et al. 2014 [2], the conjugative plasmid used as a helper and the shuttle vector should have the same OriT to improve the efficiency of conjugation.

The electroporation should not be affected by the lack of OriT and yet yielded no colonies after several attempts (with the exception of a faint colony for S. sp. PCC 6803; read more on the B12 Results page). We did not find an explanation for why it is not working since the protocol we used from Prof Hess group was said to have a high success rate (at least for PCC 6803). Possibly, a different, PCC 7942-specific electroporation protocol could be tried to further validate the shuttle vector. Also, the natural transformation did not succeed however we only tried it once (due to the time limitations) and this method of transformation has a lower efficiency, as a cyanobacteria-focused research group leader Prof. Wilde mentioned to us. Therefore, several repetitions and/or a different transformation protocol might be needed to validate the shuttle vector’s usability for natural transformation. Read more about this on the cyanobacteria B12 results page.

While searching for a reliable method to detect and measure Adenosylcobalamin (AdoCbl), alongside other techniques, we developed several sensors (BBa_K4604026, BBa_K4604027, BBa_K4604028) that can now be used by other iGEM teams. For this, we built upon other teams sensors such as Wageningen 2017. Sensors for AdoCbl in iGEM using this riboswitch up until this point had a negative signal. In our sensor the riboswitch and LacI repressor work together in such a way that a marker protein is produced in the presence of AdoCbl. This allows for extremely easy detection, since the fluorescence can even be seen by eye! Only for exact values a fluorescent measurement is needed. The marker protein can either be a fluorescent protein, like in our case sfGFP, or something different that fits other projects, which makes this sensor highly applicable to a wide range of situations. Find out more about the sensor here.