Team:LMU-Munich/Spore Coat ProteinsTour/Background

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Scientific Background of our Sporobeads

Sporobeads are Bacillus subtilis spores modulated in a way that they can be used as a platform for individual protein display. The aim of this module is to create these spores that display fusion proteins on their surface. There are several different proteins forming the spore coat layers of B. subtilis spores (Fig. 1). The outermost layer, the so-called spore crust, is composed of two proteins, CotZ and CgeA ([http://www.ncbi.nlm.nih.gov/pubmed?term=imamura%20et%20al.%202011%20spore%20crust Imamura et al., 2011]). This is why we used them to create functional fusion proteins to be expressed on our Sporobeads.

Fig. 1: Composition of the Bacillus subtilis spore coat ([http://www.ncbi.nlm.nih.gov/pubmed?term=mckenney%202010%20spore%20crust McKenney et al., 2010] & [http://www.ncbi.nlm.nih.gov/pubmed?term=imamura%20et%20al.%202011%20spore%20crust Imamura et al., 2011])

Crust Protein Genes Organization

The gene cgeA is located in the cgeABCDE cluster and is expressed from its own promoter P_cgeA. The cluster cotVWXYZ contains the gene cotZ which is cotranscribed with cotY and expressed from the promoter P_cotYZ. Another promoter of this cluster, P_cotV, is responsible for the transcription of the other three genes (Fig. 2). Those three promoters were evaluated with the lux reporter genes ([http://partsregistry.org/Part:BBa_K823025 pSB_Bs3C-luxABCDE]) to analyze their strength and the time point of their activation (see data).

Fig. 2: Gene clusters of cotZ and cgeA

Based on this data, two of the three promoters could be used for expression of spore crust fusion proteins.