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	Rhamnolipids, a class of glycolipids characterized by a rhamnose		Rhamnolipids, a class of glycolipids characterized by a rhamnose
	moiety attached to a fatty acid tail, is produced by many		moiety attached to a fatty acid tail, is produced by many
−	organism&mdash;with the <em>Pseudomonas aeruginosa</em> as the	+	organisms&mdash;with the <em>Pseudomonas aeruginosa</em> as the
	most predominate. We have shown that <em>Pseudomonas putida</em>		most predominate. We have shown that <em>Pseudomonas putida</em>
	produces both mono-rhamnolipids and di-rhamnolipids with the		produces both mono-rhamnolipids and di-rhamnolipids with the
Line 36:		Line 36:
	<p>		<p>
	Rhamnolipids are predominantly known for their biosurfactant		Rhamnolipids are predominantly known for their biosurfactant
−	properties, which possesses industrial applications	+	properties, which possesses industrial
−	<sup><a href="http://doi.org/10.1007/978-3-642-14490-5_2">8</a></sup>. Di-rhamnolipids have also been shown to repel the <em>Aedes	+	applications <sup><a href="http://doi.org/10.1007/978-3-642-14490-5_2">1</a></sup>. Di-rhamnolipids
−	aegypti</em> mosquito<sup><a href="http://doi.org/10.3389/fmicb.2015.00088">7</a></sup>. In our investigation, we have confirmed	+	have also been shown to repel the <em>Aedes aegypti</em>
−	with statistical significance that di-rhamnolipids repel Aedes	+	mosquito <sup><a href="http://doi.org/10.3389/fmicb.2015.00088">2</a></sup>. In
−	aegypti. We have also shown with statistical significance that	+	our investigation, we have confirmed with statistical significance
−	mono-rhamnolipids repel <em>Aedes aegypti</em>. The compatibility of	+	that di-rhamnolipids repel Aedes aegypti. We have also shown with
−	rhamnolipids with human skin was also a main concern of ours&mdash;as	+	statistical significance that mono-rhamnolipids repel <em>Aedes
−	rhamnolipids have been shown to be a virulence factor. We have	+	aegypti</em>. The compatibility of rhamnolipids with human skin
−	shown that rhamnolipids are compatible with human keratinocytes in	+	was also a main concern of ours&mdash;as rhamnolipids have been
−	the presence of both <em>Pseudomonas aeruginosa</em> and <em>Pseudomonas	+	shown to be a virulence factor. We have shown that rhamnolipids
−	putida</em>. Likewise, we have shown that rhamnolipids are compatible	+	are compatible with human keratinocytes in the presence of
−	with <em>Staphylococcus epidermidis</em>&mdash;a skin microbiome	+	both <em>Pseudomonas aeruginosa</em> and <em>Pseudomonas
−	organism. Lastly, we have confirmed the both mono-rhamnolipids and	+	putida</em>. Likewise, we have shown that rhamnolipids are
−	di-rhamnolipids are producible in <em>Pseudomonas putida</em> with the addition of	+	compatible with <em>Staphylococcus epidermidis</em>&mdash;a skin
−	rhlAB and rhlC, respectively.	+	microbiome organism. Lastly, we have confirmed the both
		+	mono-rhamnolipids and di-rhamnolipids are producible
		+	in <em>Pseudomonas putida</em> with the addition of rhlAB and
		+	rhlC, respectively.
		+	</p>
		+
		+	<h1>
		+	Convenient quantification of rhamnolipid（BNDS China 2021）.
		+	</h1>
		+	<p>
		+
		+	Rhamnolipid as a glycolipid, is difficult to quantify due to its lack of characteristic signals under most kinds of spectrometry. A relatively accurate way to quantify rhamnolipid is HPLC-MS, yet it’s expensive and difficult to access especially in high school and small laboratories. Here we(BNDS China 2021) documented a fast and convenient way to quantify it while still maintaining a good accuracy.
		+	<br/><br/>
		+	The oil spreading method measures the ability of a solution to lower the surface tension and emulsify mixture of hydrophobic and hydrophilic substances. The concentration of rhamnolipid can be demonstrated since its rhamnose head is polar and its lipid tail is non-polar.
		+	<br/><br/>
		+	Before measuring the rhamnolipid concentration of the sample, a standard curve should be created using the same solvent as the sample, in our case is the LB culture. Concentrations are set at 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/L. These standard samples are used to create the standard curve as shown below.
		+	</p >
		+
		+
		+	<img src="https://2021.igem.org/wiki/images/7/7c/T--BNDS_China--contrbution--Standardcurve.png" alt=""  width="700" />
		+	<i>
		+	Figure 1: the standard curve created with three replicates. (Error bars are too small to be seen for 700-900 mg/L)
		+
		+	</i>
		+
		+	From the curve we can observe significant trend between 50 mg/L – 500 mg/L, yet as the concentration increase, its difference in the diameters decrease. By using quadratic regression, we found a line of best fit with R2=0.9848. Showing that the curve y = -0.1016x2 + 1.7694x - 1.0383 is reliable. ANOVA test further supported this, the p-value between 50 – 500 mg/L are smaller than 5%, in which we can reject the null hypothesis and accept that differences are significant between these groups. However, p-value gets greater and reaches 0.8 between 500 and 600 mg/L, this implies that as the concentration increases, the results become unreliable and we cannot accurately predict the sample’s concentration using the diameters. To solve this problem, the size of the petri dish, the amount of paraffin added, and the amount of sample added can be adjusted so until the sample lies in the reliable region.
		+	<br/><br/>
		+
		+	Method:<br/><br/>
		+	1. Dissolve 2 g of Sudan II into 50 ml of liquid paraffin, mix thoroughly until it’s entirely dissolved with no visible clumps of the solute. The solution should be dark red.
		+	<br/><br/>
		+	2. Add 30 ml of deionized water into a 90mm petri dish.
		+	<br/><br/>
		+	3. Add 6 ml of the paraffin solution with pipette. Be careful so that the paraffin stays on the surface of the water and forms a thin layer that covers the entire surface. Do not let the paraffin sink to the bottom of the petri dish.
		+	<br/><br/>
		+	4. Add 30ul of the prepared sample into the center of the paraffin layer. Measure and record the diameter of the water circle the expands from the center. Do this immediately, otherwise the circle will shrink and lead to systematic error.
		+	<h1 id="putida">Mutant rhlC <em>P. putida</em> produces
		+	di-rhamnolipids</h1>
		+
		+	<h2>Quantification of rhamnolipids</h2>
		+	<p>
		+	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-C<sub>10</sub>-C<sub>10</sub>:
		+	pseudomolecular ion of 503.56 m/z) and
		+	di-rhamnolipids (rha-rha-C<sub>10</sub>-C<sub>10</sub>: 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.
	</p>		</p>
−	<h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,	+	<p>
		+	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 <em>P. putida</em> 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 &#x00b5;g/mL of
		+	rha-C<sub>10</sub>-C<sub>10</sub> and 3.524 &#x00b5;g/mL of
		+	rha-rha-C<sub>10</sub>-C<sub>10</sub> were detected.
		+	</p>
		+	<figure>
		+	<img src="https://static.igem.org/mediawiki/parts/9/9c/RhlABC1.png" alt="mono-rhamnolipids production" width="700">
		+	</figure>
		+	<figure>
		+	<img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC2.png" alt="di-rhamnolipids production" width="700">
		+	</figure>
		+
		+	<h1 id="Mosqitoes">Di-Rhamnolipids repel <em>Aedes Aegypti</em></h1>
		+	<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>
Line 74:		Line 157:
	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
Line 87:		Line 170:
	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 100:		Line 183:
	dehydrogenase enzymes, which are only active in		dehydrogenase enzymes, which are only active in
	live (metabolically active)		live (metabolically active)
−	cells<sup><a href="http://www.biovision.com/manuals/K300.pdf">6</a></sup>. For	+	cells<sup><a href="http://www.biovision.com/manuals/K300.pdf">3</a></sup>. For
	the MTS assay, pure media were used as a negative		the MTS assay, pure media were used as a negative
	control (100% cell death), and keratinocyte		control (100% cell death), and keratinocyte
Line 111:		Line 194:
	alt="Keratinocyte P. putida coculture" width="500">		alt="Keratinocyte P. putida coculture" width="500">
	</figure>		</figure>
−	<p>
−	We originally tried to do plating experiments to
−	see if keratinocytes internalized any bacteria,
−	but were unable to completely kill off all the
−	bacteria in the keratinocyte supernatant even at
−	extremely high gentamicin concentrations and thus
−	could not get an accurate read.
−	</p>
	<p>		<p>
	The results indicate that there is no consistent		The results indicate that there is no consistent
Line 141:		Line 216:
	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
Line 151:		Line 226:
	but only slowed down the growth.		but only slowed down the growth.
	</p>		</p>
−	<p>	+	<figure>
−	A cassette containing a promoter, a GFP gene, the RhlAB gene, and	+	<img src="https://static.igem.org/mediawiki/parts/3/30/Staph_rhamno.png"
−	a terminator was combined with the Staphylococcus-compatible	+	alt="S. Epidermidis Growth in the presence of rhamnolipids" width="600">
−	plasmids, pC194 and pC221, to obtain our recombinant GFP tagged	+	</figure>
−	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.	+
−	</p>	+
−	<p>	+
−	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.	+
−	</p>	+
−	<h1 id="putida">Mutant rhlC <em>P. putida</em> produces	+
−	di-rhamnolipids</h1>	+
−		+
−	<h2>Transformation of <em>P. putida</em> KT2440</h2>	+
−	<p>	+
−	In order to avoid the virulence factors of	+
−	<em>Pseudomonas aeruginosa</em>, bacterial strains with	+
−	similar or shared metabolic pathways to the one	+
−	above were chosen as potential candidates. The	+
−	final candidates were <em>Pseudomonas putida</em> and	+
−	<em>Staphylococcus epidermidis</em>. Although	+
−	<em>S. epidermidis</em> doesn’t share the same exact	+
−	pathway as <em>P. aeruginosa</em>, it is a	+
−	naturally-occurring skin microbiome and only need	+
−	two additional enzymes, RhlA and RhlB, to produce	+
−	mono-rhamnolipids, and one additional enzyme, RhlC, to convert mono-rhamnolipids to di-rhamnolipids. Genes rhlA, rhlB, and rhlC necessary	+
−	for di-rhamnolipid synthesis were extracted from	+
−	the <em>P. aeruginosa</em> 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 and 3).	+
−	<p>	+
−	In order to investigate the amount of di-rhamnolipids produced, we	+	<hr>
−	have tested our mutant strains of <em>P. putida</em> transformed	+	<p>
−	with rhlC gene. It was grown under the same condition of 24 hours	+	<sup><a href="http://doi.org/10.1007/978-3-642-14490-5_2">1</a></sup>
−	incubation in LB media supplemented by 50 g/L of	+	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.<br>
−	glucose. Approximately 142 &#x00b5;g/mL of	+	<sup><a href="http://doi.org/10.3389/fmicb.2015.00088">2</a></sup>
−	rha-C<sub>10</sub>-C<sub>10</sub> and 3.524 &#x00b5;g/mL of	+	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.<br>
−	rha-rha-C<sub>10</sub>-C<sub>10</sub> were detected.	+	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.<br>
		+	<sup><a href="http://www.biovision.com/manuals/K300.pdf">3</a></sup>
		+	"MTS Cell Proliferation Colorimetric Assay Kit."
		+	BioVision. Web.
	</p>		</p>
	</html>		</html>
		+

---

Latest revision as of 05:34, 21 October 2021

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.

Convenient quantification of rhamnolipid（BNDS China 2021）.

Rhamnolipid as a glycolipid, is difficult to quantify due to its lack of characteristic signals under most kinds of spectrometry. A relatively accurate way to quantify rhamnolipid is HPLC-MS, yet it’s expensive and difficult to access especially in high school and small laboratories. Here we(BNDS China 2021) documented a fast and convenient way to quantify it while still maintaining a good accuracy.

The oil spreading method measures the ability of a solution to lower the surface tension and emulsify mixture of hydrophobic and hydrophilic substances. The concentration of rhamnolipid can be demonstrated since its rhamnose head is polar and its lipid tail is non-polar.

Before measuring the rhamnolipid concentration of the sample, a standard curve should be created using the same solvent as the sample, in our case is the LB culture. Concentrations are set at 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/L. These standard samples are used to create the standard curve as shown below.

Figure 1: the standard curve created with three replicates. (Error bars are too small to be seen for 700-900 mg/L) From the curve we can observe significant trend between 50 mg/L – 500 mg/L, yet as the concentration increase, its difference in the diameters decrease. By using quadratic regression, we found a line of best fit with R2=0.9848. Showing that the curve y = -0.1016x2 + 1.7694x - 1.0383 is reliable. ANOVA test further supported this, the p-value between 50 – 500 mg/L are smaller than 5%, in which we can reject the null hypothesis and accept that differences are significant between these groups. However, p-value gets greater and reaches 0.8 between 500 and 600 mg/L, this implies that as the concentration increases, the results become unreliable and we cannot accurately predict the sample’s concentration using the diameters. To solve this problem, the size of the petri dish, the amount of paraffin added, and the amount of sample added can be adjusted so until the sample lies in the reliable region.

Method:

1. Dissolve 2 g of Sudan II into 50 ml of liquid paraffin, mix thoroughly until it’s entirely dissolved with no visible clumps of the solute. The solution should be dark red.

2. Add 30 ml of deionized water into a 90mm petri dish.

3. Add 6 ml of the paraffin solution with pipette. Be careful so that the paraffin stays on the surface of the water and forms a thin layer that covers the entire surface. Do not let the paraffin sink to the bottom of the petri dish.

4. Add 30ul of the prepared sample into the center of the paraffin layer. Measure and record the diameter of the water circle the expands from the center. Do this immediately, otherwise the circle will shrink and lead to systematic error.

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