Characterization of rhlAB
The RhlAB operon codes for mono-rhamnolipid, a virulence factor naturally found in the Pseudomonas aeruginosa. It contains the RhlA and RhlB genes within the operon, coding for the final two enzymes in the mono-rhamnolipid production pathway. Rhamnolipids, consisting of a rhamnose sugar attached to one or multiple lipid side chains, has many uses and has been highlighted by previous iGEM teams—such as Panama iGEM 2010—as a biosurfactant capable of aiding oil recovery.
Furthermore, according to the article, "Rhamnolipids: solution against Aedes Aegypti?" published by Universidade Estadual Paulista Júlio de Mesquita Filho Rio Claro in Brazil, this biosurfactant has a capability of repelling mosquitoes as well1. Using this information, we have decided to create a microorganism that secretes rhamnolipids constitutively and can be applied to human skin with no safety hazards.
Although a BioBrick for RhlAB has been previously documented by Team Panama in 2011, there was no substantial characterization on this part. Furthermore, its ability to repel mosquitoes hasn't been documented or characterized. Our team this year, not only performed an accurate quantitative analysis of the gene expression in E. Coli, but also characterized in a different chassis, Pseudomonas putida KT2440. In addition, in order to quantify how effectively rhamnolipids repel mosquitoes, we conducted mosquito feeding and landing assays. Finally, we also tested the level of toxicity of RhlAB on a skin microbiome, specifically, Staphylococcus epidermidis.
Mosquito landing and feeding assay
Aedes aegypti, the species of mosquito observed to carry Zika virus, were grown from larval stage, and females were sorted at the pupae or adult stage. Since only females consume blood for reproduction, we were only interested in using them for the assays.
One day before experiment, 50 mosquitos, containing 30 females, were isolated in cages and starved from 23-25 hours. Each cage was then taken to a warm room (~30 oC), and the cage was covered with wet paper towels to preserve humidity. For each trial, our blood feeding system (Figure 1) was then placed on top of the cage each with a different cotton gauze soaked with negative control water, 1 mg/mL mono-rhamnolipid solution, 1 mg/mL di-rhamnolipid solution, or positive control 25% DEET, and the mosquito activity was videotaped for 1 hour. Afterwards, the cage was taken to the cold room to paralyze the assayed mosquitoes, and mosquitoes that had consumed blood were counted. Our results showed that while DEET was the strongest mosquito repellent with no landings or fed mosquitos, 1 mg/mL mono and di-rhamnolipid still showed significant repulsion as shown in the graph below.
Characterization of E. coli and P. putida
Because our team's aim was to create a "live mosquito repellent" bacteria that constitutively produced rhamnolipids, we focused on transforming two organisms: Pseudomonas putida and Staphylococcus epidermidis. Along the way, we encountered many issues, suggesting that high amounts of rhamnolipid may pose a metabolic burden or be toxic to these species. At the end, however, we successfully electroporated our RhlAB construct with a "low expression level" promoter into P. putida.
In order to accurately measure the amount of rhamnolipids produced by our mutant strains, we used supercritical fluid chromatography (SFC-MS). First, a test run was executed with a mixture of mono-rhamnolipids and di-rhamnolipids at the concentration of 5 mg/mL by running the sample through the column packed with 2-PIC. From this test run, we have obtained the retention times of mono-rhamnolipids (rha-C10-C10: pseudomolecular ion of 503.56 m/z) and di-rhamnolipids (rha-rha-C10-C10: pseudomolecular ion of 649.8 m/z) to be approximately 3.974 min and 4.942 min respectively. Then, a calibration curve was constructed with 95% pure mono-rhamnolipids, and the limit of detection was found to be approximately 5 ug/mL. The mass fractions were obtained from electrospray ionization (ESI) negative mode.
From our TLC analysis, it was found that supplementing the LB media with glucose is crucial to the production of rhamnolipid. Therefore, for SFC-MS analysis, all the mutant strains (E. Coli_RhlAB, E. Coli_L1_RhlAB, and P. Putida_L1_RhlAB) were grown in LB supplemented with glucose. From the SFC-MS data, it was found that mutant E. Coli strain makes more mono-rhamnolipids than mutant P. putida. Furthermore, the promoter strength was confirmed as expected since the mutant E. coli strain transformed with a high expression level promoter H2 produced almost 6 times more rha-C10-C10.
Cloning rhlAB into skin microbiome and skin bacteria toxicity assays
Since Staphylococcus species, especially S. epidermidis, are not commonly cloned, the cloning process was quite challenging; more details can be seen under our protocol section. While we were unable to clone RhlAB into Staphylococcus epidermidis, were we able to clone it into the shuttle species Staphylococcus aureus. While S. Aureus is pathogenic and cannot be used as a commensal skin bacteria, our ability to clone RhlAB into it suggests that it is possible for RhlAB to be produced in epidermidis. Because the Staphylococcus species have never been cloned to produce rhamnolipids, and rhamnolipids have been characterized as antibiotic, we tested the effect of different concentrations of rhamnolipids (2000 mg to 15.6 mg per liter) on Staphylococcal growth.
1 Silva, Vinicius L., Roberta B. Lovaglio, Claudio J. Von Zuben, and Jonas Contiero. “Rhamnolipids: solution against Aedes Aegypti?” Frontiers in Microbiology. 6.88 (2015) Web.