Revision as of 05:18, 24 October 2016 (view source) KJung (Talk | contribs) m ← Older edit		Revision as of 05:22, 24 October 2016 (view source) KJung (Talk | contribs) m Newer edit →
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	</p>		</p>
	<figure>		<figure>
−	<img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC1.png" alt="E. coli" width="700">	+	<img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC1.png" alt="mono-rhamnolipids production" width="700">
	</figure>		</figure>
	<figure>		<figure>
−	<img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC2.png" alt="E. coli" width="700">	+	<img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC2.png" alt="di-rhamnolipids production" width="700">
	</figure>		</figure>
−	</p>	+	<h1 id="Mosqitoes">Di-Rhamnolipids repel <em>Aedes Aegypti</em></h1>
−	<h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,	+	<p>
		+	In order to quantify how effectively rhamnolipids repel mosquitoes, we conducted mosquito feeding and landing assays. <em>Aedes aegypti</em>, 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.
		+	</p>
		+	<p>
		+	One day before experiment, 50 total mosquitos (with 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) was placed on top of the cage each with a cotton gauze soaked with either 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.  It is important to note that the age of female mosquitoes and the time of feeding played an important role in how mosquitoes behave.  Typically, it is optimum to use female mosquitoes of age from 4-6 days for feeding assays as any mosquitoes older than this age range will be too old to reproduce, and thereby not needing to drink blood.  Furthermore, their feeding is most active 4 hours before dusk.  Some of our trials that didn’t meet these criteria did not result in any feeding, but we did observe significant difference in landing between the control and rhamnolipids.  Our landing assay 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 statistically significant repulsion as shown in the graph below.
		+	</p>
		+	<figure>
		+	<img src="https://static.igem.org/mediawiki/parts/8/8a/Mosquito.png"
		+	alt="Mosquito Experiment" width="650">
		+	</figure>
		+	<h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
	and rhamnolipids are compatible with human		and rhamnolipids are compatible with human
	keratinocytes</h1>		keratinocytes</h1>
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	concentration of rhamnolipid that induces 50% cell		concentration of rhamnolipid that induces 50% cell
	death. The IC50 was determined to be between 45.19		death. The IC50 was determined to be between 45.19
−	&#x00b5;/mL and 65.52 &#x00b5;/mL. Relating the results to	+	&#x00b5;g/mL and 65.52 &#x00b5;g/mL. Relating the results to
	rhamnolipid quantification, the concentration of		rhamnolipid quantification, the concentration of
	rhamnolipid the construct produces should not		rhamnolipid the construct produces should not
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	Keratinocytes were co-cultured with different		Keratinocytes were co-cultured with different
	strains of bacteria (<em>Pseudomonas putida</em>,		strains of bacteria (<em>Pseudomonas putida</em>,
−	<em>Pseudomonas aeruginosa PAK</em>, <em>Staphylococcus aureus</em>,	+	<em>Pseudomonas aeruginosa</em> PAK, <em>Staphylococcus aureus</em>,
	<em>Staphylococcus epidermidis</em>, and mutant rhlAB		<em>Staphylococcus epidermidis</em>, and mutant rhlAB
	<em>P. putida</em>). Half were cultured in plain DMEM with		<em>P. putida</em>). Half were cultured in plain DMEM with
Line 175:		Line 185:
	epidermidis</em></h1>		epidermidis</em></h1>
	<p>		<p>
−	In order to make sure that our S. Aureus strain (RN4220) and our	+	In order to make sure that our <em>S. aureus</em> strain (RN4220) and our
−	S. Epidermidis (RP62A, 1457) strains would not be killed by the	+	<em>S. epidermidis</em> (RP62A, 1457) strains would not be killed by the
	production of rhamnolipids, we conducted 3 rhamnolipid survival		production of rhamnolipids, we conducted 3 rhamnolipid survival
	assays with the 1g/L rhamnolipids necessary for mosquito		assays with the 1g/L rhamnolipids necessary for mosquito
−	repelling.  Kanamycin added to S. Epidermidis cell culture was	+	repelling.  Kanamycin added to <em>S. epidermidis</em> cell culture was
	used as a negative control.  Although the addition of higher		used as a negative control.  Although the addition of higher
	concentrations of rhamnolipids (250 mg/L and above) depressed the		concentrations of rhamnolipids (250 mg/L and above) depressed the
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	but only slowed down the growth.		but only slowed down the growth.
	</p>		</p>
		+	<figure>
		+	<img src="https://static.igem.org/mediawiki/parts/3/30/Staph_rhamno.png"
		+	alt="S. Epidermidis Growth in the presence of rhamnolipids" width="600">
		+	</figure>

---

Revision as of 05:22, 24 October 2016

rhamnosyltransferase 2 [Pseudomonas aeruginosa]

Rhamnolipids, a class of glycolipids characterized by a rhamnose moiety attached to a fatty acid tail, is produced by many organisms—with the Pseudomonas aeruginosa as the most predominate. We have shown that Pseudomonas putida produces both mono-rhamnolipids and di-rhamnolipids with the addition of the rhlAB and rhlC operons, respectively. Previous research has shown that di-rhamnolipids repel the Aedes aegypti mosquito. We have shown that both di-rhamnolipids and mono-rhamnolipids repel Aedes aegypti. We have also shown that rhamnolipids are compatible with human keratinocytes in the presence of both Pseudomonas aeruginosa and Pseudomonas putida. Lastly, we have shown that rhamnolipids are compatible with Staphylococcus epidermidis—a skin microbiome organism.

Introduction

Rhamnolipids are a class of glycolipids characterized by a rhamnose moiety and a fatty acid tail. While rhamnolipids are produced in a variety of organisms, Pseudomonas aeruginosa is most frequently cited. In Pseudomonas aeruginosa, genes rhlA and rhlB are cooperative to from the complex rhlAB that codes for the enzyme rhamnosyltransferase 1. The enzyme rhamnosyltransferase 1 catalyzes the addition of a (hydroxyalkanoyloxy)alkanoic acid (HAA) fatty acid tail to a rhamnose sugar to produce a mono-rhamnolipid. Similarly, rhlC codes for the enzyme rhamnosyltransferase 2, which catalyzes an addition of another rhamnose moiety to a mono-rhamnolipid to form a di-rhamnolipid.

Rhamnolipids are predominantly known for their biosurfactant properties, which possesses industrial applications ¹. Di-rhamnolipids have also been shown to repel the Aedes aegypti mosquito ². In our investigation, we have confirmed with statistical significance that di-rhamnolipids repel Aedes aegypti. We have also shown with statistical significance that mono-rhamnolipids repel Aedes aegypti. The compatibility of rhamnolipids with human skin was also a main concern of ours—as rhamnolipids have been shown to be a virulence factor. We have shown that rhamnolipids are compatible with human keratinocytes in the presence of both Pseudomonas aeruginosa and Pseudomonas putida. Likewise, we have shown that rhamnolipids are compatible with Staphylococcus epidermidis—a skin microbiome organism. Lastly, we have confirmed the both mono-rhamnolipids and di-rhamnolipids are producible in Pseudomonas putida with the addition of rhlAB and rhlC, respectively.

Mutant rhlC P. putida produces di-rhamnolipids

Quantification of rhamnolipids

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. 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, our mutant strain P. putida RhlABC was grown in LB supplemented with 50 g/L of glucose for 24 hours. From the SFC-MS data, it was found that approximately 142 µg/mL of rha-C₁₀-C₁₀ and 3.524 µg/mL of rha-rha-C₁₀-C₁₀ were detected.

Di-Rhamnolipids repel Aedes Aegypti

In order to quantify how effectively rhamnolipids repel mosquitoes, we conducted mosquito feeding and landing assays. 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 total mosquitos (with 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) was placed on top of the cage each with a cotton gauze soaked with either 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. It is important to note that the age of female mosquitoes and the time of feeding played an important role in how mosquitoes behave. Typically, it is optimum to use female mosquitoes of age from 4-6 days for feeding assays as any mosquitoes older than this age range will be too old to reproduce, and thereby not needing to drink blood. Furthermore, their feeding is most active 4 hours before dusk. Some of our trials that didn’t meet these criteria did not result in any feeding, but we did observe significant difference in landing between the control and rhamnolipids. Our landing assay 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 statistically significant repulsion as shown in the graph below.

P. putida, S. epidermidis, and rhamnolipids are compatible with human keratinocytes

Determination of rhamnolipid IC50

Keratinocytes, human skin cells, were grown for several days. When the cells were 80% confluent, they were seeded in 24 well plates at a density of 2.5105. The cells were weaned off of antibiotics the following day before they were treated with varying concentrations of rhamnolipids and the reagent MTS. The MTS assay reveals the cell viability of the cells. Using this information, the data was normalized and statistically analyzed to determine the keratinocyte IC50—or the concentration of rhamnolipid that induces 50% cell death. The IC50 was determined to be between 45.19 µg/mL and 65.52 µg/mL. Relating the results to rhamnolipid quantification, the concentration of rhamnolipid the construct produces should not cause significant cell death.

Keratinocyte cell viability bacteria assay

Keratinocytes were co-cultured with different strains of bacteria (Pseudomonas putida, Pseudomonas aeruginosa PAK, Staphylococcus aureus, Staphylococcus epidermidis, and mutant rhlAB P. putida). Half were cultured in plain DMEM with serum, and half were culture in DMEM with 1 mg/mL mixed mono- and di- rhamnolipids. After co-culturing, the keratinocytes were washed with PBS, exposed to gentamicin in an attempt to kill the bacteria, and incubated in MTS cell viability assay for up to 4 hours and viewed in a plate reader. MTS assay is colorimetric cell viability assay and reacts with NADPH-dependent dehydrogenase enzymes, which are only active in live (metabolically active) cells³. For the MTS assay, pure media were used as a negative control (100% cell death), and keratinocyte culture with normal DMEM was used as a positive control (“0%” cell death, or the maximum number of cells that could be alive).

The results indicate that there is no consistent trend regarding the addition of rhamnolipid and cell viability. Rhamnolipids did not significantly increase or decrease cell viability regardless of the bacteria type as shown in the first figure since the error bars overlap. We hypothesized that the concentration of P. putida would not influence cell viability as it is an environmental strain not nearly as potent as other bacterial strains such as Pseudomonas aeruginosa PAK. As depicted in the second figure, all MOIs (ranging from 0 to 20) did not significantly influence the cell viability of the strain as shown by the overlapping error bars in the graph. These results overall indicate that our construct may not cause significant cell death once applied to the skin in an acute setting of a few hours.

Rhamnolipids are compatible with 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.

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¹ Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.
² Silva, Vinicius L., Roberta B. Lovaglio, Claudio J. Zuben, and Jonas Contiero. "Rhamnolipids: Solution against Aedes Aegypti?" Frontiers. Frontiers in Microbiology, 16 Feb. 2015. Web. 23 Oct. 2016.
Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.
³ "MTS Cell Proliferation Colorimetric Assay Kit." BioVision. Web.

Sequence and Features