The work with Clostridia

Despite the great opportunities offered by the Clostridium genus, only a few teams have worked with it so far. Most teams (e.g., Aachen 2019) have focused on the few pathogenic Clostridia, particularly Clostridium difficile. However, this does not represent the biotechnological opportunities the Clostridium class can provide. Only iGEM Nottingham and iGEM UNAM-Mexico 2011 took the first cloning steps within this bacterial genus. As Clostridia are providing great biotechnological relevance and can contribute immensely to a carbon-neutral future, we took steps to establish this bacterial class in the iGEM community.
As only scarce information on the handling of Clostridia and the cloning system was provided by iGEM Nottingham 2018/2019, we aimed to expand these results and provide a detailed guide on how to work with anaerobic Clostridia, which pitfalls need to be overcome, and what is to consider when planning a project involving these bacteria.
We created a detailed guide on which equipment is required for cultivation, which molecular tools can be applied, and what to consider when applying these tools. To make this clostridial class more accessible for future iGEM teams, we focus on widely established molecular tools to provide a solid base that can later be expanded. Therefore, we aim to make Clostridia widely accessible and open up this high-potential bacterial group to the iGEM Community. For more information, please have a look at our Clostrida Guide.

In addition, we have expanded obatined knowledge on the ClosTron plasmid and the thiolase, ferredoxin, and galactose-induced promoter introduced by iGEM Nottingham:

Approaching our goal

For the expression of genes of the carbon assimilation pathway (Wood-Ljungdahl pathway) in our target Clostridium, we have built on the knowledge gathered by the iGEM Team Nottingham 2019, who introduced the ClosTron system (Figure 1) into the iGEM community which was originally published by Heap et al. (2009). This plasmid system provides the great advantage that it can be introduced into aerobic E. coli and anaerobic Clostridia. As we aimed to introduce large and potentially toxic gene constructs to this plasmid system, we expanded the knowledge by new low plasmid copy systems and new cultivation strategies for difficult constructs. We combined different plasmids with clostridium-specific replication origins and non-clostridium replication origins with varying promoter systems. For the final gene expression, we had two targets. First: Introduce the gene of interest into E. coli and the Clostridium to check for toxicity and plasmid amplification. For this purpose, we choose the pbgal promoter as this acts as an inducible promoter and was shown by iGEM Nottingham to have the lowest expression strength. For the final expression of the pathway and to obtain high yields, a promoter with strong expression strength is desired. For this purpose, we chose the thiolase promoter of iGEM Nottingham as this was shown to be one of the strongest promoters.

Contributions

Image and ideas from ​​​​Heap, J. T., Pennington, O. J., Cartman, S. T., & Minton, N. P. (2009). A modular system for Clostridium shuttle plasmids. Journal of Microbiological Methods, 78(1), 79–85., doi:10.1016/j.mimet.2009.05.004