Rhamnolipids are naturally synthesized by the skin bacteria Pseudomonas aeruginosa using the metabolic pathway illustrated in Figure 1.
In order to avoid the virulence factors of Pseudomonas aeruginosa, bacterial strains with similar or shared metabolic pathways to the one above were chosen as potential candidates. The final candidates were Pseudomonas putida and Staphylococcus epidermidis. Although S. epidermidis doesn't share the same exact pathway as P. aeruginosa, it is a naturally-occurring skin microbiome and only need two additional enzymes, RhlA and RhlB, to produce mono-rhamnolipids. rhlA and rhlB genes necessary for mono-rhamnolipid synthesis were extracted from the P. aeruginosa P14 bacterial strain to be placed into the modified plasmid pNJ3.1 for transformation into the desired bacterial strains (Figure 2). The plasmid pC194 and a shuttle vector strain, S. aureus RN4220 (details on S. Epidermidis transformation are discussed in the experiments and result section) were used for S. epidermidis transformations with the same basic design (Figure 2). The conversion of mono-rhamnolipids to di-rhamnolipids requires the additional gene rhlC, which was also extracted from P14 strain and cloned into the same pNJ3.1 vector (Figure 2).
The plasmid pNJ3.1 has a promoter library that includes hundreds of constitutive promoters with the length of 180 base pairs taken from various microbiome. It is located at the upstream of the gene that codes for super-folded GFP, and the expression level of each constitutive promoter is quantified with the intensity of fluorescence excited at 480 nm and emitted at 511 nm.