Part:BBa_K2062006:Design
rhamnosyltransferase 2 [Pseudomonas aeruginosa]
Rhamnolipids are naturally synthesized by the skin bacteria Pseudomonas aeruginosa using the metabolic pathway illustrated in Figure 1.
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. 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, were used for S. epidermidis transformations with the same basic design (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.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 622
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 205
Illegal NgoMIV site found at 393
Illegal NgoMIV site found at 931 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 664
Illegal BsaI.rc site found at 898