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	in <em>Pseudomonas putida</em> with the addition of rhlAB and		in <em>Pseudomonas putida</em> with the addition of rhlAB and
	rhlC, respectively.		rhlC, respectively.
		+	</p>
		+	<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>
		+	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 glucose. From the SFC-MS data, it was found
		+	that the mutant strain when transformed with rhlAB and rhlC gene
		+	</p>
		+	<figure>
		+	<img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC1.png" alt="E. coli" width="700">
		+	</figure>
		+	<figure>
		+	<img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC2.png" alt="E. coli" width="700">
		+	</figure>
		+
		+	<p>
		+	We have also tested the mutant strain of <em>S. aureus</em>
		+	RN4220, the strain that carries shuttle vector for
		+	<em>S. epidermidis</em>. Unfortunately, SFC-MS data didn't
		+	show any production of rhamnolipids from <em>S. aureus</em>
		+	strain.
		+	</p>
		+	<figure>
		+	<img src="https://static.igem.org/mediawiki/2016/a/a3/Ecoli-sfc-2.png" alt="E. coli" width="700">
		+	</figure>
		+	<p>
		+	In order to investigate the amount of di-rhamnolipids produced, we
		+	have tested our mutant strains of <em>P. putida</em> transformed
		+	with rhlC gene. It was grown under the same condition of 24 hours
		+	incubation in LB media supplemented by 50 g/L of
		+	glucose. Approximately 142 &#x00b5;/mL of
		+	rha-C<sub>10</sub>-C<sub>10</sub> and 3.524 &#x00b5;/mL of
		+	rha-rha-C<sub>10</sub>-C<sub>10</sub> were detected.
		+	</p>
		+
	</p>		</p>
	<h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,		<h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
Line 153:		Line 210:
	growth of all our Staphylococcal species, it didn’t kill the cells		growth of all our Staphylococcal species, it didn’t kill the cells
	but only slowed down the growth.		but only slowed down the growth.
−	</p>
−	<p>
−	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.
	</p>		</p>
	<p>		<p>
Line 187:		Line 218:
	S. epidermidis 1457.		S. epidermidis 1457.
	</p>		</p>
−	<h1 id="putida">Mutant rhlAB <em>P. putida</em> produces	+
−	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. Genes rhlA and rhlB necessary	+
−	for mono-rhamnolipid synthesis were extracted from	+
−	the <em>P. aeruginosa P14</em> 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). The	+
−	plasmid pC194 and a shuttle vector strain,	+
−	<em>S. aureus</em> RN4220 (details on <em>S. epidermidis</em>	+
−	transformation are discussed in the experiments	+
−	and result section) were used for <em>S. epidermidis</em>	+
−	transformations with the same basic design (Figure	+
−	3). The conversion of mono-rhamnolipids to	+
−	di-rhamnolipids requires the additional gene rhlC,	+
−	which was also extracted from P14 strain and	+
−	cloned into the same pNJ3.1 vector (Figure 4).	+
−	</p>	+
−	<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. Then, a calibration	+
−	curve was constructed with 95% pure	+
−	mono-rhamnolipids, and the limit of detection was	+
−	found to be approximately 5 &#x00b5;/mL. The mass	+
−	fractions were obtained from electrospray	+
−	ionization (ESI) negative mode.	+
−	</p>	+
−	<figure>	+
−	<img src="https://static.igem.org/mediawiki/2016/2/29/Pputida-sfc.png" alt="P. putida" width="700">	+
−	</figure>	+
−	<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, 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 <em>E. coli</em> strain makes more	+
−	mono-rhamnolipids than mutant	+
−	<em>P. putida</em>. Furthermore, the promoter strength was	+
−	confirmed as expected since the mutant <em>E. coli</em>	+
−	strain transformed with a high expression level	+
−	promoter H2 produced almost 6 times more	+
−	rha-C<sub>10</sub>-C<sub>10</sub>.	+
−	</p>	+
−	<figure>	+
−	<img src="https://static.igem.org/mediawiki/2016/a/a5/Ecoli-sfc.png" alt="E. coli" width="700">	+
−	</figure>	+
−	<p>	+
−	In order to investigate the optimum growth	+
−	conditions for rhamnolipid by the mutant <em>P. putida</em>	+
−	strain, the amount of glucose added and the time	+
−	of growth were varied. Using the calibration curve	+
−	above, we were able to measure the accurate amount	+
−	of rhamnolipids produced in each cell	+
−	culture. From this data, we have concluded that	+
−	<em>P. putida</em> produces the most mono-rhamnolipids when	+
−	grown for 24 hours in the media LB supplemented	+
−	with 50 g/L of glucose.	+
−	</p>	+
−	<p>	+
−	We have also tested the mutant strain of <em>S. aureus</em>	+
−	RN4220, the strain that carries shuttle vector for	+
−	<em>S. epidermidis</em>. Unfortunately, SFC-MS data didn't	+
−	show any production of rhamnolipids from <em>S. aureus</em>	+
−	strain.	+
−	</p>	+
−	<figure>	+
−	<img src="https://static.igem.org/mediawiki/2016/a/a3/Ecoli-sfc-2.png" alt="E. coli" width="700">	+
−	</figure>	+
−	<p>	+
−	In order to investigate the amount of di-rhamnolipids produced, we	+
−	have tested our mutant strains of <em>P. putida</em> transformed	+
−	with rhlC gene. It was grown under the same condition of 24 hours	+
−	incubation in LB media supplemented by 50 g/L of	+
−	glucose. Approximately 142 &#x00b5;/mL of	+
−	rha-C<sub>10</sub>-C<sub>10</sub> and 3.524 &#x00b5;/mL of	+
−	rha-rha-C<sub>10</sub>-C<sub>10</sub> were detected.	+
−	</p>	+
	<hr>		<hr>
	<p>		<p>

---

Revision as of 05:04, 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 glucose. From the SFC-MS data, it was found that the mutant strain when transformed with rhlAB and rhlC gene

We have also tested the mutant strain of S. aureus RN4220, the strain that carries shuttle vector for S. epidermidis. Unfortunately, SFC-MS data didn't show any production of rhamnolipids from S. aureus strain.

In order to investigate the amount of di-rhamnolipids produced, we have tested our mutant strains of P. putida transformed with rhlC gene. It was grown under the same condition of 24 hours incubation in LB media supplemented by 50 g/L of glucose. Approximately 142 µ/mL of rha-C₁₀-C₁₀ and 3.524 µ/mL of rha-rha-C₁₀-C₁₀ were detected.

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 µ/mL and 65.52 µ/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).

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.

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.

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.

---

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