Difference between revisions of "Part:BBa K2062005:Design"

Latest revision as of 20:07, 25 October 2016

Rhamnosyltransferase 1 [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 3).

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.

Transformation of Staphylococcus epidermidis

In order to make sure that our S. aureus strain (RN4220) and our S. Epidermidis (RP62A, 1457) strains would not be killed by the production of rhamnolipids, we conducted 3 rhamnolipid survival assays with the 1g/L rhamnolipids necessary for mosquito repelling. Kanamycin added to S. Epidermidis cell culture was used as a negative control. Although the addition of higher concentrations of rhamnolipids (250 mg/L and above) depressed the growth of all our Staphylococcal species, it didn’t kill the cells but only slowed down the growth.

A cassette containing a promoter, a GFP gene, the rhlAB gene, and a terminator was combined with the Staphylococcus-compatible plasmids, pC194 and pC221, to obtain our recombinant GFP tagged rhamnolipid plasmid. There are 2 schemes we used for Staphylococcus transformation: electroporation and conjugation. For electroporation, S. aureus RN4220 and S. aureus OS2 were electroporated with dialyzed pC194_H1_RhlAB. Only S. aureus OS2 had any GFP positive colonies, and DNA from the GFP positive OS2 was then dialyzed for electroporation into S. epidermidis RP62A. However, even after repetitions of this procedure, the transformed strain of S. epidermidis did not produce any GFP positive colonies. For conjugation, OS2/pGO1 was first electroporated with pC221_RhlAB H1, M3, and L1. Only pC221_L1_RhlAB produced colonies that had the correct band size of 3300 base pairs, but these colonies were not GFP positive. Then, OS2/pGO1 with the RhlAB gene was combined with S. epidermidis RP62A on a 0.45um Millipore filter placed on a BHI agar plate. Despite our repeated effort, this procedure did not produce any GFP positive colonies. In an attempt to overcome a possible restriction enzyme activity in S. epidermidis, we tried the heat inactivation for host restriction system described by Lofblom et al. 2006. in Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism. However, that did not seem to help either.

As an alternative system, we tried transforming a vector from E. Coli methyltransferase deficient into S. epidermidis. While we got our recombinant pC194_RhlAB of all promoter strengths into the E. coli, we were unable to electroporate our construct into S. epidermidis 1457.

Sequence and Features