Difference between revisions of "Part:BBa K2062006"
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                <h2 id="mosquitos">Rhamnolipids repel <em>Aedes aegypti</em> mosquitos</h2>
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  <h1>Introduction</h1>
−
 
+
  <p>
−
<p>
+
    Rhamnolipids are a class of glycolipids characterized by a
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                    In order to quantify how effectively rhamnolipids
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    rhamnose moiety and a fatty acid tail. While rhamnolipids are
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                    repel mosquitoes, we conducted mosquito feeding
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    produced in a variety of organisms, <em>Pseudomonas aeruginosa</em> is most
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                    and landing assays. Aedes aegypti, the species of
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    frequently cited. In <em>Pseudomonas aeruginosa</em>, genes rhlA and rhlB
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                    mosquito observed to carry Zika virus, were grown
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    are cooperative to from the complex rhlAB that codes for the
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                    from larval stage, and females were sorted at the
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    enzyme rhamnosyltransferase 1. The enzyme rhamnosyltransferase 1
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                    pupae or adult stage. Since only females consume
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    catalyzes the addition of a (hydroxyalkanoyloxy)alkanoic acid
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                    blood for reproduction, we were only interested in
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    (HAA) fatty acid tail to a rhamnose sugar to produce a
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                    using them for the assays.
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    mono-rhamnolipid. Similarly, rhlC codes for the enzyme
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                  [[File:Mosquito-1.png|frame|test]]
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    rhamnosyltransferase 2, which catalyzes an addition of another
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</p>
+
    rhamnose moiety to a mono-rhamnolipid to form a di-rhamnolipid.
−
 
+
  </p>
−
 
+
  <p>
−
 
+
    Rhamnolipids are predominantly known for their biosurfactant
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<figure>
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    properties, which possesses industrial applications
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  <img src="https://static.igem.org/mediawiki/2016/b/b0/Mosquito-2.png"
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    (cite). Di-rhamnolipids have also been shown to repel the <em>Aedes
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      alt="Cage Setup" width="360">
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      aegypti</em> mosquito (cite). In our investigation, we have confirmed
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</figure>
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    with statistical significance that di-rhamnolipids repel Aedes
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                <p>
+
    aegypti. We have also shown with statistical significance that
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                    One day before experiment, 50 total mosquitos
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    mono-rhamnolipids repel <em>Aedes aegypti</em>. The compatibility of
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                    (with 30 females) were isolated in cages and
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    rhamnolipids with human skin was also a main concern of ours&mdash;as
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                    starved from 23-25 hours. Each cage was then taken
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    rhamnolipids have been shown to be a virulence factor. We have
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                    to a warm room (~30 &deg;C), and the cage was covered
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    shown that rhamnolipids are compatible with human keratinocytes in
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                    with wet paper towels to preserve humidity. For
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    the presence of both <em>Pseudomonas aeruginosa</em> and <em>Pseudomonas
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                    each trial, our blood feeding system (Figure) was
+
      putida</em>. Likewise, we have shown that rhamnolipids are compatible
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                    placed on top of the cage each with a cotton gauze
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    with <em>Staphylococcus epidermidis</em>&mdash;a skin microbiome
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                    soaked with either negative control water, 1 mg/mL
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    organism. Lastly, we have confirmed the both mono-rhamnolipids and
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                    mono-rhamnolipid solution, 1 mg/mL di-rhamnolipid
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    di-rhamnolipids are producible in <em>Pseudomonas putida</em> with the addition of
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                    solution, or positive control 25% DEET, and the
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    rhlAB and rhlC, respectively.
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                    mosquito activity was videotaped for 1
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  </p>
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                    hour. Afterwards, the cage was taken to the cold
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  <h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
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                    room to paralyze the assayed mosquitoes, and
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    and rhamnolipids are compatible with human
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                    mosquitoes that had consumed blood were
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    keratinocytes</h1>
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                    counted. It is important to note that the age of
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  <hr>
−
                    female mosquitoes and the time of feeding played
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  <h2>Determination of rhamnolipid IC50</h2>
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                    an important role in how mosquitoes
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  <figure>
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                    behave. Typically, it is optimum to use female
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    <img src="https://static.igem.org/mediawiki/2016/b/b7/Keratinocyte-rhamnolipid-ic50.png"
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                    mosquitoes of age from 4-6 days for feeding assays
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alt="Keratinocyte IC50" width="800">
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                    as any mosquitoes older than this age range will
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  </figure>
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                    be too old to reproduce, and thereby not needing
+
  <p>
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                    to drink blood. Furthermore, their feeding is most
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    Keratinocytes, human skin cells, were grown for
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                    active 4 hours before dusk. Some of our trials
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    several days. When the cells were 80% confluent,
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                    that didn’t meet these criteria did not result in
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    they were seeded in 24 well plates at a density of
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                    any feeding, but we did observe significant
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    2.5105. The cells were weaned off of antibiotics
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                    difference in landing between the control and
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    the following day before they were treated with
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                    rhamnolipids. Our landing assay results showed
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    varying concentrations of rhamnolipids and the
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                    that while DEET was the strongest mosquito
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    reagent MTS. The MTS assay reveals the cell
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                    repellent with no landings or fed mosquitos, 1
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    viability of the cells. Using this information,
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                    mg/mL mono and di-rhamnolipid still showed
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    the data was normalized and statistically analyzed
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                    statistically significant repulsion as shown in
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    to determine the keratinocyte IC50&mdash;or the
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                    the graph below.
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    concentration of rhamnolipid that induces 50% cell
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                </p>
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    death. The IC50 was determined to be between 45.19
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<figure>
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    &#x00b5;/mL and 65.52 &#x00b5;/mL. Relating the results to
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  <img src="https://static.igem.org/mediawiki/2016/1/17/Mosquito-landing.png"
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    rhamnolipid quantification, the concentration of
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      alt="Mosquito Landing" width="500">
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    rhamnolipid the construct produces should not
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</figure>
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    cause significant cell death.
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                <h2 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
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  </p>
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      and rhamnolipids are compatible with human
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  <h2>Keratinocyte cell viability bacteria assay</h2>
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      keratinocytes</h2>
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  <figure>
−
                <hr>
+
    <img src="https://static.igem.org/mediawiki/2016/5/58/Keratinocyte-species.jpg"
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                <h3>Determination of rhamnolipid IC50</h3>
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alt="Keratinocyte species">
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<figure>
+
  </figure>
−
  <img src="https://static.igem.org/mediawiki/2016/b/b7/Keratinocyte-rhamnolipid-ic50.png"
+
  <p>
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      alt="Keratinocyte IC50" width="800">
+
    Keratinocytes were co-cultured with different
−
</figure>
+
    strains of bacteria (<em>Pseudomonas putida</em>,
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                <p>
+
    <em>Pseudomonas aeruginosa PAK</em>, <em>Staphylococcus aureus</em>,
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                    Keratinocytes, human skin cells, were grown for
+
    <em>Staphylococcus epidermidis</em>, and mutant rhlAB
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                    several days. When the cells were 80% confluent,
+
    <em>P. putida</em>). Half were cultured in plain DMEM with
−
                    they were seeded in 24 well plates at a density of
+
    serum, and half were culture in DMEM with 1 mg/mL
−
                    2.5105. The cells were weaned off of antibiotics
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    mixed mono- and di- rhamnolipids. After
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                    the following day before they were treated with
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    co-culturing, the keratinocytes were washed with
−
                    varying concentrations of rhamnolipids and the
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    PBS, exposed to gentamicin in an attempt to kill
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                    reagent MTS. The MTS assay reveals the cell
+
    the bacteria, and incubated in MTS cell viability
−
                    viability of the cells. Using this information,
+
    assay for up to 4 hours and viewed in a plate
−
                    the data was normalized and statistically analyzed
+
    reader. MTS assay is colorimetric cell viability
−
                    to determine the keratinocyte IC50&mdash;or the
+
    assay and reacts with NADPH-dependent
−
                    concentration of rhamnolipid that induces 50% cell
+
    dehydrogenase enzymes, which are only active in
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                    death. The IC50 was determined to be between 45.19
+
    live (metabolically active)
−
                    &#x00b5;/mL and 65.52 &#x00b5;/mL. Relating the results to
+
    cells<sup><a href="http://www.biovision.com/manuals/K300.pdf">6</a></sup>. For
−
                    rhamnolipid quantification, the concentration of
+
    the MTS assay, pure media were used as a negative
−
                    rhamnolipid the construct produces should not
+
    control (100% cell death), and keratinocyte
−
                    cause significant cell death.
+
    culture with normal DMEM was used as a positive
−
                </p>
+
    control (“0%” cell death, or the maximum number of
−
                <h3>Keratinocyte cell viability bacteria assay</h3>
+
    cells that could be alive).
−
<figure>
+
  </p>
−
  <img src="https://static.igem.org/mediawiki/2016/5/58/Keratinocyte-species.jpg"
+
  <figure>
−
      alt="Keratinocyte species">
+
    <img src="https://static.igem.org/mediawiki/2016/6/6a/Keratinocyte-putida.png"
−
</figure>
+
alt="Keratinocyte P. putida coculture" width="500">
−
                <p>
+
  </figure>
−
                    Keratinocytes were co-cultured with different
+
  <p>
−
                    strains of bacteria (<em>Pseudomonas putida</em>,
+
    We originally tried to do plating experiments to
−
                    <em>Pseudomonas aeruginosa PAK</em>, <em>Staphylococcus aureus</em>,
+
    see if keratinocytes internalized any bacteria,
−
                    <em>Staphylococcus epidermidis</em>, and mutant rhlAB
+
    but were unable to completely kill off all the
−
                    <em>P. putida</em>). Half were cultured in plain DMEM with
+
    bacteria in the keratinocyte supernatant even at
−
                    serum, and half were culture in DMEM with 1 mg/mL
+
    extremely high gentamicin concentrations and thus
−
                    mixed mono- and di- rhamnolipids. After
+
    could not get an accurate read.
−
                    co-culturing, the keratinocytes were washed with
+
  </p>
−
                    PBS, exposed to gentamicin in an attempt to kill
+
  <p>
−
                    the bacteria, and incubated in MTS cell viability
+
    The results indicate that there is no consistent
−
                    assay for up to 4 hours and viewed in a plate
+
    trend regarding the addition of rhamnolipid and
−
                    reader. MTS assay is colorimetric cell viability
+
    cell viability. Rhamnolipids did not significantly
−
                    assay and reacts with NADPH-dependent
+
    increase or decrease cell viability regardless of
−
                    dehydrogenase enzymes, which are only active in
+
    the bacteria type as shown in the first figure
−
                    live (metabolically active)
+
    since the error bars overlap. We hypothesized that
−
                    cells<sup><a href="http://www.biovision.com/manuals/K300.pdf">6</a></sup>. For
+
    the concentration of <em>P. putida</em> would not influence
−
                    the MTS assay, pure media were used as a negative
+
    cell viability as it is an environmental strain
−
                    control (100% cell death), and keratinocyte
+
    not nearly as potent as other bacterial strains
−
                    culture with normal DMEM was used as a positive
+
    such as <em>Pseudomonas aeruginosa PAK</em>. As depicted in
−
                    control (“0%” cell death, or the maximum number of
+
    the second figure, all MOIs (ranging from 0 to 20)
−
                    cells that could be alive).
+
    did not significantly influence the cell viability
−
                </p>
+
    of the strain as shown by the overlapping error
−
<figure>
+
    bars in the graph. These results overall indicate
−
  <img src="https://static.igem.org/mediawiki/2016/6/6a/Keratinocyte-putida.png"
+
    that our construct may not cause significant cell
−
      alt="Keratinocyte P. putida coculture" width="500">
+
    death once applied to the skin in an acute setting
−
</figure>
+
    of a few hours.
−
                <p>
+
  </p>
−
                    We originally tried to do plating experiments to
+
  <h1 id="putida">Mutant rhlAB <em>P. putida</em>
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                    see if keratinocytes internalized any bacteria,
+
    produces rhamnolipids</h1>
−
                    but were unable to completely kill off all the
+
  <hr>
−
                    bacteria in the keratinocyte supernatant even at
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  <h2>Transformation of <em>P. putida</em> KT2440</h2>
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                    extremely high gentamicin concentrations and thus
+
  <p>
−
                    could not get an accurate read.
+
    In order to avoid the virulence factors of
−
                </p>
+
    <em>Pseudomonas aeruginosa</em>, bacterial strains with
−
                <p>
+
    similar or shared metabolic pathways to the one
−
                    The results indicate that there is no consistent
+
    above were chosen as potential candidates. The
−
                    trend regarding the addition of rhamnolipid and
+
    final candidates were <em>Pseudomonas putida</em> and
−
                    cell viability. Rhamnolipids did not significantly
+
    <em>Staphylococcus epidermidis</em>. Although
−
                    increase or decrease cell viability regardless of
+
    <em>S. epidermidis</em> doesn’t share the same exact
−
                    the bacteria type as shown in the first figure
+
    pathway as <em>P. aeruginosa</em>, it is a
−
                    since the error bars overlap. We hypothesized that
+
    naturally-occurring skin microbiome and only need
−
                    the concentration of <em>P. putida</em> would not influence
+
    two additional enzymes, RhlA and RhlB, to produce
−
                    cell viability as it is an environmental strain
+
    mono-rhamnolipids. Genes rhlA and rhlB necessary
−
                    not nearly as potent as other bacterial strains
+
    for mono-rhamnolipid synthesis were extracted from
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                    such as <em>Pseudomonas aeruginosa PAK</em>. As depicted in
+
    the <em>P. aeruginosa P14</em> bacterial strain. These
−
                    the second figure, all MOIs (ranging from 0 to 20)
+
    genes were cloned into the modified plasmid pNJ3.1
−
                    did not significantly influence the cell viability
+
    using standard cloning methods for transformation
−
                    of the strain as shown by the overlapping error
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    into the desired bacterial strains (Figure 2). The
−
                    bars in the graph. These results overall indicate
+
    plasmid pC194 and a shuttle vector strain,
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                    that our construct may not cause significant cell
+
    <em>S. aureus</em> RN4220 (details on <em>S. epidermidis</em>
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                    death once applied to the skin in an acute setting
+
    transformation are discussed in the experiments
−
                    of a few hours.
+
    and result section) were used for <em>S. epidermidis</em>
−
                </p>
+
    transformations with the same basic design (Figure
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                <h2 id="putida">Mutant rhlAB <em>P. putida</em>
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    3). The conversion of mono-rhamnolipids to
−
      produces rhamnolipids</h2>
+
    di-rhamnolipids requires the additional gene rhlC,
−
                <hr>
+
    which was also extracted from P14 strain and
−
                <h3>Transformation of <em>P. putida</em> KT2440</h3>
+
    cloned into the same pNJ3.1 vector (Figure 4).
−
                <p>
+
  </p>
−
                    In order to avoid the virulence factors of
+
  <h2>Quantification of rhamnolipids</h2>
−
                    <em>Pseudomonas aeruginosa</em>, bacterial strains with
+
  <p>
−
                    similar or shared metabolic pathways to the one
+
    In order to accurately measure the amount of
−
                    above were chosen as potential candidates. The
+
    rhamnolipids produced by our mutant strains, we
−
                    final candidates were <em>Pseudomonas putida</em> and
+
    used supercritical fluid chromatography
−
                    <em>Staphylococcus epidermidis</em>. Although
+
    (SFC-MS). First, a test run was executed with a
−
                    <em>S. epidermidis</em> doesn’t share the same exact
+
    mixture of mono-rhamnolipids and di-rhamnolipids
−
                    pathway as <em>P. aeruginosa</em>, it is a
+
    at the concentration of 5 mg/mL by running the
−
                    naturally-occurring skin microbiome and only need
+
    sample through the column packed with 2-PIC. From
−
                    two additional enzymes, RhlA and RhlB, to produce
+
    this test run, we have obtained the retention
−
                    mono-rhamnolipids. Genes rhlA and rhlB necessary
+
    times of mono-rhamnolipids (rha-C<sub>10</sub>-C<sub>10</sub>:
−
                    for mono-rhamnolipid synthesis were extracted from
+
    pseudomolecular ion of 503.56 m/z) and
−
                    the <em>P. aeruginosa P14</em> bacterial strain. These
+
    di-rhamnolipids (rha-rha-C<sub>10</sub>-C<sub>10</sub>: pseudomolecular
−
                    genes were cloned into the modified plasmid pNJ3.1
+
    ion of 649.8 m/z) to be approximately 3.974 min
−
                    using standard cloning methods for transformation
+
    and 4.942 min respectively. Then, a calibration
−
                    into the desired bacterial strains (Figure 2). The
+
    curve was constructed with 95% pure
−
                    plasmid pC194 and a shuttle vector strain,
+
    mono-rhamnolipids, and the limit of detection was
−
                    <em>S. aureus</em> RN4220 (details on <em>S. epidermidis</em>
+
    found to be approximately 5 &#x00b5;/mL. The mass
−
                    transformation are discussed in the experiments
+
    fractions were obtained from electrospray
−
                    and result section) were used for <em>S. epidermidis</em>
+
    ionization (ESI) negative mode.
−
                    transformations with the same basic design (Figure
+
  </p>
−
                    3). The conversion of mono-rhamnolipids to
+
  <figure>
−
                    di-rhamnolipids requires the additional gene rhlC,
+
    <img src="https://static.igem.org/mediawiki/2016/2/29/Pputida-sfc.png" alt="P. putida" width="700">
−
                    which was also extracted from P14 strain and
+
  </figure>
−
                    cloned into the same pNJ3.1 vector (Figure 4).
+
  <p>
−
                </p>
+
    From our TLC analysis, it was found that
−
                <h3>Quantification of rhamnolipids</h3>
+
    supplementing the LB media with glucose is crucial
−
                <p>
+
    to the production of rhamnolipid. Therefore, for
−
                    To confirm the presence of rhamnolipids produced
+
    SFC-MS analysis, all the mutant strains
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                    by our mutant strains (<em>P. putida</em>, <em>E. coli</em>
+
    (E. Coli_RhlAB, E. Coli_L1_RhlAB, and
−
                    transformed with pNJ3.1_rhlAB), we explored three
+
    P. putida_L1_RhlAB) were grown in LB supplemented
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                    different methods: cetyl trimethylammonium bromide
+
    with glucose. From the SFC-MS data, it was found
−
                    agar plating (CTAB), thin-layer chromatography
+
    that mutant <em>E. coli</em> strain makes more
−
                    (TLC), and supercritical fluid chromatography mass
+
    mono-rhamnolipids than mutant
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                    spectrometry (SFC-MS). For TLC and SFC-MS
+
    <em>P. putida</em>. Furthermore, the promoter strength was
−
                    analysis, rhamnolipids were extracted from cell
+
    confirmed as expected since the mutant <em>E. coli</em>
−
                    culture supernatant through liquid-liquid
+
    strain transformed with a high expression level
−
                    extraction with ethyl acetate and redissolved in
+
    promoter H2 produced almost 6 times more
−
                    methanol prior to measurement. Detailed protocols
+
    rha-C<sub>10</sub>-C<sub>10</sub>.
−
                    on the extraction is discussed under protocols.
+
  </p>
−
                </p>
+
  <figure>
−
                <h4>Cetyl trimethylammonium bromide agar plate assay</h4>
+
    <img src="https://static.igem.org/mediawiki/2016/a/a5/Ecoli-sfc.png" alt="E. coli" width="700">
−
<figure>
+
  </figure>
−
  <img src="https://static.igem.org/mediawiki/2016/2/29/Ctab.jpg"
+
  <p>
−
      alt="CTAB" width="300">
+
    In order to investigate the optimum growth
−
</figure>
+
    conditions for rhamnolipid by the mutant <em>P. putida</em>
−
                <p>
+
    strain, the amount of glucose added and the time
−
                  Cetyl trimethylammonium bromide (CTAB) agar plates
+
    of growth were varied. Using the calibration curve
−
                  detect the presence of rhamnolipid by reacting with
+
    above, we were able to measure the accurate amount
−
                  the sugar in
+
    of rhamnolipids produced in each cell
−
                  rhamnolipids<sup><a href="http://doi.org/10.1007/s10529-009-0049-7">7</a></sup>. When
+
    culture. From this data, we have concluded that
−
                  rhamnolipid is present, it forms blue halos around
+
    <em>P. putida</em> produces the most mono-rhamnolipids when
−
                  the compound, and the halo size usually correlates
+
    grown for 24 hours in the media LB supplemented
−
                  to the amount of
+
    with 50 g/L of glucose.
−
                  rhamnolipids<sup><a href="http://doi.org/10.1007/s10529-009-0049-7">7</a></sup>. We
+
  </p>
−
                  tested this method with 95% pure rhamnolipids
+
  <p>
−
                  (Sigma-Aldrich) by plating different concentrations
+
    We have also tested the mutant strain of <em>S. aureus</em>
−
                  of the compound dissolved in water onto SW agar
+
    RN4220, the strain that carries shuttle vector for
−
                  plate*. Blue halos were present after incubating the
+
    <em>S. epidermidis</em>. Unfortunately, SFC-MS data didn't
−
                  plate for 24 hours at 37&deg;C, but the limit of
+
    show any production of rhamnolipids from <em>S. aureus</em>
−
                  detection was too high (~1g/L). Furthermore,
+
    strain.
−
                  depending on the amount of CTAB used per plate, the
+
  </p>
−
                  size of halos varied, which made it difficult for us
+
  <figure>
−
                  to use this method as a quantitative measurement.
+
    <img src="https://static.igem.org/mediawiki/2016/a/a3/Ecoli-sfc-2.png" alt="E. coli" width="700">
−
                </p>
+
  </figure>
−
                <h4>Thin-layer chromatography</h4>
+
  <p>
−
                <p>
+
    In order to investigate the amount of
−
                    Thin-layer chromatography (TLC) was used as a more
+
    di-rhamnolipids produced, we have tested our
−
                    reliable method of detecting rhamnolipids. TLC is
+
    mutant strains of <em>P. putida</em> transformed with rhlC
−
                    a very common separation technique used to isolate
+
    gene. It was grown under the same condition of 24
−
                    a desired compound from a mixture. It typically
+
    hours incubation in LB media supplemented by 50
−
                    involves two different phases, stationary and
+
    g/L of glucose. Approximately 142 &#x00b5;/mL of
−
                    mobile, in which the mobile phase flows through
+
    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>
−
                    the stationary phase and carries the components of
+
    were detected.
−
                    the mixture with
+
  </p>
−
                    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>
 
</html>
  

Revision as of 00:39, 24 October 2016


rhamnosyltransferase 2 [Pseudomonas aeruginosa]

RhlC codes for rhamnosyltransferase 2, which is the last enzyme in the rhamnolipid production pathway. Its function is to convert mono-rhamnolipid to di-rhamnolipid by catalyzing an additional dTDP-L-rhamnose.(1) It was cloned from Pseudomonas aeruginosa PAO1 from an operon also containing a putative fosfomycin resistance protein and putative MFS transporter(2).

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.

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