Part:BBa_K2062005:Experience

Transformation of P. putida KT2440

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. Genes rhlA and rhlB necessary for mono-rhamnolipid synthesis were extracted from the P. aeruginosa P14 bacterial strain. These genes were cloned into the modified plasmid pNJ3.1 using standard cloning methods 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 3). 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 4).

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alt="RhlAB Plasmid Circuit" width="500">

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