Difference between revisions of "Part:BBa K2062005"

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The RhlAB operon codes for mono-rhamnolipid, a glycolipid naturally produced by <i>Pseudomonas aeruginosa</i>. Our part 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, have many uses and have been highlighted by previous iGEM teams as a biosurfactant capable of aiding oil recovery [1]. Another recently shown novel use of rhamnolipids is its mosquito repellent properties. Silva et al recently showed that 1 mg/mL mixed mono- and di-rhamnolipids (harvested from <i>P. aeruginosa</i>) has the ability to repel Aedes aegypti mosquito, the biggest known vector for Zika virus and a carrier of many other tropical diseases.[2] We replicated Silva et. al’s study with mono-rhamnolipid, and our results indicate that mono-rhamnolipid repelled 70% of mosquitoes (see graph below).
 
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<html>
 
<html>
−
                <h2 id="mosquitos">Rhamnolipids repel <em>Aedes aegypti</em> mosquitos</h2>
+
  <p>
 +
    Rhamnolipids, a class of glycolipids characterized by a rhamnose
 +
    moiety attached to a fatty acid tail, is produced by many
 +
    organism&mdash;with the <em>Pseudomonas aeruginosa</em> as the
 +
    most predominate. We have shown that <em>Pseudomonas putida</em>
 +
    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 <em>Aedes aegypti</em>. We have also shown
 +
    that rhamnolipids are compatible with human keratinocytes in the
 +
    presence of both <em>Pseudomonas aeruginosa</em>
 +
    and <em>Pseudomonas putida</em>. Lastly, we have shown that
 +
    rhamnolipids are compatible with <em>Staphylococcus
 +
    epidermidis</em>&mdash;a skin microbiome organism.
 +
  </p>
 +
  <h1>Introduction</h1>
 +
  <p>
 +
    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, <em>Pseudomonas aeruginosa</em> is most
 +
    frequently cited. In <em>Pseudomonas aeruginosa</em>, 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.
 +
  </p>
 +
  <p>
 +
    Rhamnolipids are predominantly known for their biosurfactant
 +
    properties, which possesses industrial applications
 +
    (cite). Di-rhamnolipids have also been shown to repel the <em>Aedes
 +
      aegypti</em> mosquito (cite). 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 <em>Aedes aegypti</em>. The compatibility of
 +
    rhamnolipids with human skin was also a main concern of ours&mdash;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 <em>Pseudomonas aeruginosa</em> and <em>Pseudomonas
 +
      putida</em>. Likewise, we have shown that rhamnolipids are compatible
 +
    with <em>Staphylococcus epidermidis</em>&mdash;a skin 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 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
 +
    and rhamnolipids are compatible with human
 +
    keratinocytes</h1>
 +
 
 +
  <h2>Determination of rhamnolipid IC50</h2>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/2016/b/b7/Keratinocyte-rhamnolipid-ic50.png"
 +
alt="Keratinocyte IC50" width="800">
 +
  </figure>
 +
  <p>
 +
    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&mdash;or the
 +
    concentration of rhamnolipid that induces 50% cell
 +
    death. The IC50 was determined to be between 45.19
 +
    &#x00b5;/mL and 65.52 &#x00b5;/mL. Relating the results to
 +
    rhamnolipid quantification, the concentration of
 +
    rhamnolipid the construct produces should not
 +
    cause significant cell death.
 +
  </p>
 +
  <h2>Keratinocyte cell viability bacteria assay</h2>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/2016/5/58/Keratinocyte-species.jpg"
 +
alt="Keratinocyte species">
 +
  </figure>
 +
  <p>
 +
    Keratinocytes were co-cultured with different
 +
    strains of bacteria (<em>Pseudomonas putida</em>,
 +
    <em>Pseudomonas aeruginosa PAK</em>, <em>Staphylococcus aureus</em>,
 +
    <em>Staphylococcus epidermidis</em>, and mutant rhlAB
 +
    <em>P. putida</em>). 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)
 +
    cells<sup><a href="http://www.biovision.com/manuals/K300.pdf">6</a></sup>. 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).
 +
  </p>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/2016/6/6a/Keratinocyte-putida.png"
 +
alt="Keratinocyte P. putida coculture" width="500">
 +
  </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>
 +
    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 <em>P. putida</em> would not influence
 +
    cell viability as it is an environmental strain
 +
    not nearly as potent as other bacterial strains
 +
    such as <em>Pseudomonas aeruginosa PAK</em>. 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.
 +
  </p>
 +
  <h1>Rhamnolipids are compatible with <em>Staphylococcus
 +
      epidermidis</em></h1>
 +
  <p>
 +
    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.
 +
  </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>
 +
    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 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>
 +
</html>
  
−
<p>
 
−
                    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.
 
−
                  [[File:Mosquito-1.png|frame|test]]
 
−
</p>
 
−
 
−
 
−
 
−
<figure>
 
−
  <img src="https://static.igem.org/mediawiki/2016/b/b0/Mosquito-2.png"
 
−
      alt="Cage Setup" width="360">
 
−
</figure>
 
−
                <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 &deg;C), 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/2016/1/17/Mosquito-landing.png"
 
−
      alt="Mosquito Landing" width="500">
 
−
</figure>
 
−
                <h2 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
 
−
      and rhamnolipids are compatible with human
 
−
      keratinocytes</h2>
 
−
                <hr>
 
−
                <h3>Determination of rhamnolipid IC50</h3>
 
−
<figure>
 
−
  <img src="https://static.igem.org/mediawiki/2016/b/b7/Keratinocyte-rhamnolipid-ic50.png"
 
−
      alt="Keratinocyte IC50" width="800">
 
−
</figure>
 
−
                <p>
 
−
                    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&mdash;or the
 
−
                    concentration of rhamnolipid that induces 50% cell
 
−
                    death. The IC50 was determined to be between 45.19
 
−
                    &#x00b5;/mL and 65.52 &#x00b5;/mL. Relating the results to
 
−
                    rhamnolipid quantification, the concentration of
 
−
                    rhamnolipid the construct produces should not
 
−
                    cause significant cell death.
 
−
                </p>
 
−
                <h3>Keratinocyte cell viability bacteria assay</h3>
 
−
<figure>
 
−
  <img src="https://static.igem.org/mediawiki/2016/5/58/Keratinocyte-species.jpg"
 
−
      alt="Keratinocyte species">
 
−
</figure>
 
−
                <p>
 
−
                    Keratinocytes were co-cultured with different
 
−
                    strains of bacteria (<em>Pseudomonas putida</em>,
 
−
                    <em>Pseudomonas aeruginosa PAK</em>, <em>Staphylococcus aureus</em>,
 
−
                    <em>Staphylococcus epidermidis</em>, and mutant rhlAB
 
−
                    <em>P. putida</em>). 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)
 
−
                    cells<sup><a href="http://www.biovision.com/manuals/K300.pdf">6</a></sup>. 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).
 
−
                </p>
 
−
<figure>
 
−
  <img src="https://static.igem.org/mediawiki/2016/6/6a/Keratinocyte-putida.png"
 
−
      alt="Keratinocyte P. putida coculture" width="500">
 
−
</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>
 
−
                    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 <em>P. putida</em> would not influence
 
−
                    cell viability as it is an environmental strain
 
−
                    not nearly as potent as other bacterial strains
 
−
                    such as <em>Pseudomonas aeruginosa PAK</em>. 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.
 
−
                </p>
 
−
                <h2 id="putida">Mutant rhlAB <em>P. putida</em>
 
−
      produces rhamnolipids</h2>
 
−
                <hr>
 
−
                <h3>Transformation of <em>P. putida</em> KT2440</h3>
 
−
                <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>
 
−
                <h3>Quantification of rhamnolipids</h3>
 
−
                <p>
 
−
                    To confirm the presence of rhamnolipids produced
 
−
                    by our mutant strains (<em>P. putida</em>, <em>E. coli</em>
 
−
                    transformed with pNJ3.1_rhlAB), we explored three
 
−
                    different methods: cetyl trimethylammonium bromide
 
−
                    agar plating (CTAB), thin-layer chromatography
 
−
                    (TLC), and supercritical fluid chromatography mass
 
−
                    spectrometry (SFC-MS). For TLC and SFC-MS
 
−
                    analysis, rhamnolipids were extracted from cell
 
−
                    culture supernatant through liquid-liquid
 
−
                    extraction with ethyl acetate and redissolved in
 
−
                    methanol prior to measurement. Detailed protocols
 
−
                    on the extraction is discussed under protocols.
 
−
                </p>
 
−
                <h4>Cetyl trimethylammonium bromide agar plate assay</h4>
 
−
<figure>
 
−
  <img src="https://static.igem.org/mediawiki/2016/2/29/Ctab.jpg"
 
−
      alt="CTAB" width="300">
 
−
</figure>
 
−
                <p>
 
−
                  Cetyl trimethylammonium bromide (CTAB) agar plates
 
−
                  detect the presence of rhamnolipid by reacting with
 
−
                  the sugar in
 
−
                  rhamnolipids<sup><a href="http://doi.org/10.1007/s10529-009-0049-7">7</a></sup>. When
 
−
                  rhamnolipid is present, it forms blue halos around
 
−
                  the compound, and the halo size usually correlates
 
−
                  to the amount of
 
−
                  rhamnolipids<sup><a href="http://doi.org/10.1007/s10529-009-0049-7">7</a></sup>. We
 
−
                  tested this method with 95% pure rhamnolipids
 
−
                  (Sigma-Aldrich) by plating different concentrations
 
−
                  of the compound dissolved in water onto SW agar
 
−
                  plate*. Blue halos were present after incubating the
 
−
                  plate for 24 hours at 37&deg;C, but the limit of
 
−
                  detection was too high (~1g/L). Furthermore,
 
−
                  depending on the amount of CTAB used per plate, the
 
−
                  size of halos varied, which made it difficult for us
 
−
                  to use this method as a quantitative measurement.
 
−
                </p>
 
−
                <h4>Thin-layer chromatography</h4>
 
−
                <p>
 
−
                    Thin-layer chromatography (TLC) was used as a more
 
−
                    reliable method of detecting rhamnolipids. TLC is
 
−
                    a very common separation technique used to isolate
 
−
                    a desired compound from a mixture. It typically
 
−
                    involves two different phases, stationary and
 
−
                    mobile, in which the mobile phase flows through
 
−
                    the stationary phase and carries the components of
 
−
                    the mixture with
 
−
                    it<sup><a href="http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html">8</a></sup>. Separation
 
−
                    of compounds is based on the affinity of the
 
−
                    compound towards the stationary phase vs. the
 
−
                    mobile phase, and depending on which phase the
 
−
                    compounds prefer, they travel with the solvent at
 
−
                    different
 
−
                    rates<sup><a href="http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html">8</a></sup>. We
 
−
                    used silica gel as the stationary phase and
 
−
                    solvent consisted of chloroform, methanol, and
 
−
                    acetic acid in 65:15:2 % volume ratio as the
 
−
                    mobile phase. Knowing that di-rhamnolipids have
 
−
                    more hydroxyl groups, we predicted it to have a
 
−
                    smaller retention factor than mono-rhamnolipids as
 
−
                    they would prefer to stay on polar silica gel. To
 
−
                    visualize the plate, the silica gel plate was
 
−
                    stained with four different dyes: CAM, KMnO4,
 
−
                    orcinol with 50% H2SO4, and orcinol with 10%
 
−
                    H2SO4. Among the four staining methods, orcinol
 
−
                    with 10% gave the best visibility. The chemical
 
−
                    mechanism in which orcinol and sulfuric acid react
 
−
                    with rhamnose to create a dye is illustrated in
 
−
                    Figure 2.
 
−
                </p>
 
−
<figure>
 
−
  <img src="https://static.igem.org/mediawiki/2016/8/84/Tlc-multiple.png" alt="TLC" width="600">
 
−
</figure>
 
−
                <p>
 
−
                    We confirmed that TLC method shows two distinct
 
−
                    bands for mono-rhamnolipids and di-rhamnolipids,
 
−
                    and that it has a limit of detection lower than
 
−
                    CTAB (approximately 0.5 mg/mL). Next, we tested
 
−
                    our mutant <em>P. putida</em> and <em>E. coli</em> strains with
 
−
                    promoters of different strengths. For positive
 
−
                    controls, WT <em>P. aeruginosa</em> and mutant
 
−
                    <em>P. aeruginosa</em> were used, and for a negative
 
−
                    control, WT <em>P. putida</em> was tested. When the cells
 
−
                    were grown in LB only media, none of the
 
−
                    rhamnolipids was detected from <em>P. putida</em> or
 
−
                    <em>E. coli</em>. However, when the cells were grown in LB
 
−
                    supplemented with glucose, a faint band for
 
−
                    mono-rhamnolipids was detected from mutant <em>E. coli</em>
 
−
                    transformed with a high expression level
 
−
                    promoter. Although our construct in <em>P. putida</em>
 
−
                    didn’t show any clear band, mutant <em>P. aeruginosa</em>
 
−
                    transformed with the same construct showed to
 
−
                    produce a lot more mono-rhamnolipids compared to
 
−
                    WT <em>P. aeruginosa</em>, which mainly produces
 
−
                    di-rhamnolipids. This result confirms that our
 
−
                    construct is working as expected, yet we need a
 
−
                    detection method with higher sensitivity.
 
−
                </p>
 
−
<figure>
 
−
  <img src="https://static.igem.org/mediawiki/2016/4/49/Tlc-2.png" alt="TLC" width="500">
 
−
</figure>
 
−
                <h4>Supercritical fluid chromatography</h4>
 
−
                <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>
 
−
</html>
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 01:03, 24 October 2016


Rhamnosyltransferase 1 [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 (cite). Di-rhamnolipids have also been shown to repel the Aedes aegypti mosquito (cite). 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) cells6. 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.


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