Difference between revisions of "Part:BBa K2062006"
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>
 
   </p>
 
   <h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
 
   <h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
Line 100: Line 103:
 
     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 184: Line 187:
 
     S. epidermidis 1457.
 
     S. epidermidis 1457.
 
   </p>
 
   </p>
−
   <h1 id="putida">Mutant rhlC <em>P. putida</em> produces
+
   <h1 id="putida">Mutant rhlAB <em>P. putida</em> produces
−
     di-rhamnolipids</h1>
+
     rhamnolipids</h1>
 
    
 
    
 
   <h2>Transformation of <em>P. putida</em> KT2440</h2>
 
   <h2>Transformation of <em>P. putida</em> KT2440</h2>
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     naturally-occurring skin microbiome and only need
 
     naturally-occurring skin microbiome and only need
 
     two additional enzymes, RhlA and RhlB, to produce
 
     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
+
     mono-rhamnolipids. Genes rhlA and rhlB necessary
−
     for di-rhamnolipid synthesis were extracted from
+
     for mono-rhamnolipid synthesis were extracted from
−
     the <em>P. aeruginosa</em> P14 bacterial strain. These
+
     the <em>P. aeruginosa P14</em> bacterial strain. These
 
     genes were cloned into the modified plasmid pNJ3.1
 
     genes were cloned into the modified plasmid pNJ3.1
 
     using standard cloning methods for transformation
 
     using standard cloning methods for transformation
−
     into the desired bacterial strains (Figure 2 and 3).  
+
     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>
 
   <p>
 
     In order to investigate the amount of di-rhamnolipids produced, we
 
     In order to investigate the amount of di-rhamnolipids produced, we
Line 211: Line 288:
 
     with rhlC gene. It was grown under the same condition of 24 hours
 
     with rhlC gene. It was grown under the same condition of 24 hours
 
     incubation in LB media supplemented by 50 g/L of
 
     incubation in LB media supplemented by 50 g/L of
−
     glucose. Approximately 142 &#x00b5;g/mL of
+
     glucose. Approximately 142 &#x00b5;/mL of
−
     rha-C<sub>10</sub>-C<sub>10</sub> and 3.524 &#x00b5;g/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.
 
     rha-rha-C<sub>10</sub>-C<sub>10</sub> were detected.
 +
  </p>
 +
  <hr>
 +
  <p>
 +
    <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>
 +
  
  

Revision as of 01:57, 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 organism—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.

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

Determination of rhamnolipid IC50

Keratinocyte 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

Keratinocyte species

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).

Keratinocyte P. putida coculture

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.

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.

Mutant rhlAB P. putida produces rhamnolipids

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. Genes rhlA and rhlB necessary for mono-rhamnolipid synthesis were extracted from the P. aeruginosa 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). The plasmid pC194 and a shuttle vector strain, S. aureus RN4220 (details on S. epidermidis transformation are discussed in the experiments and result section) were used for S. epidermidis 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).

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. Then, a calibration curve was constructed with 95% pure mono-rhamnolipids, and the limit of detection was found to be approximately 5 µ/mL. The mass fractions were obtained from electrospray ionization (ESI) negative mode.

P. putida

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.

E. coli

In order to investigate the optimum growth conditions for rhamnolipid by the mutant P. putida 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 P. putida produces the most mono-rhamnolipids when grown for 24 hours in the media LB supplemented with 50 g/L of glucose.

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.

E. coli

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.

3 "MTS Cell Proliferation Colorimetric Assay Kit." BioVision. Web.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 622
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 205
    Illegal NgoMIV site found at 393
    Illegal NgoMIV site found at 931
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 664
    Illegal BsaI.rc site found at 898