Difference between revisions of "Part:BBa K2062006"
 
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<partinfo>BBa_K2062006 short</partinfo>
 
<partinfo>BBa_K2062006 short</partinfo>
  
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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 <em>Pseudomonas aeruginosa</em> PAO1 from an operon also containing a putative fosfomycin resistance protein and putative MFS transporter(2).  
+
<html>
 +
  <p>
 +
    Rhamnolipids, a class of glycolipids characterized by a rhamnose
 +
    moiety attached to a fatty acid tail, is produced by many
 +
    organisms&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 <sup><a href="http://doi.org/10.1007/978-3-642-14490-5_2">1</a></sup>. Di-rhamnolipids
 +
    have also been shown to repel the <em>Aedes aegypti</em>
 +
    mosquito <sup><a href="http://doi.org/10.3389/fmicb.2015.00088">2</a></sup>. 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>
  
−
                <h2 id="mosquitos">Rhamnolipids repel <em>Aedes aegypti</em> mosquitos</h2>
+
  <h1>
 +
                            Convenient quantification of rhamnolipid（BNDS China 2021）.
 +
    </h1>
 +
    <p>
 +
       
 +
        Rhamnolipid as a glycolipid, is difficult to quantify due to its lack of characteristic signals under most kinds of spectrometry. A relatively accurate way to quantify rhamnolipid is HPLC-MS, yet it’s expensive and difficult to access especially in high school and small laboratories. Here we(BNDS China 2021) documented a fast and convenient way to quantify it while still maintaining a good accuracy.
 +
    <br/><br/>
 +
The oil spreading method measures the ability of a solution to lower the surface tension and emulsify mixture of hydrophobic and hydrophilic substances. The concentration of rhamnolipid can be demonstrated since its rhamnose head is polar and its lipid tail is non-polar.
 +
<br/><br/>
 +
Before measuring the rhamnolipid concentration of the sample, a standard curve should be created using the same solvent as the sample, in our case is the LB culture. Concentrations are set at 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/L. These standard samples are used to create the standard curve as shown below.
 +
</p >
  
 +
 +
    <img src="https://2021.igem.org/wiki/images/7/7c/T--BNDS_China--contrbution--Standardcurve.png" alt=""  width="700" />
 +
    <i>
 +
                        Figure 1: the standard curve created with three replicates. (Error bars are too small to be seen for 700-900 mg/L)
 +
 +
    </i>
 +
 +
From the curve we can observe significant trend between 50 mg/L – 500 mg/L, yet as the concentration increase, its difference in the diameters decrease. By using quadratic regression, we found a line of best fit with R2=0.9848. Showing that the curve y = -0.1016x2 + 1.7694x - 1.0383 is reliable. ANOVA test further supported this, the p-value between 50 – 500 mg/L are smaller than 5%, in which we can reject the null hypothesis and accept that differences are significant between these groups. However, p-value gets greater and reaches 0.8 between 500 and 600 mg/L, this implies that as the concentration increases, the results become unreliable and we cannot accurately predict the sample’s concentration using the diameters. To solve this problem, the size of the petri dish, the amount of paraffin added, and the amount of sample added can be adjusted so until the sample lies in the reliable region. 
 +
<br/><br/>
 +
 +
Method:<br/><br/>
 +
1. Dissolve 2 g of Sudan II into 50 ml of liquid paraffin, mix thoroughly until it’s entirely dissolved with no visible clumps of the solute. The solution should be dark red.
 +
<br/><br/>
 +
2. Add 30 ml of deionized water into a 90mm petri dish.
 +
<br/><br/>
 +
3. Add 6 ml of the paraffin solution with pipette. Be careful so that the paraffin stays on the surface of the water and forms a thin layer that covers the entire surface. Do not let the paraffin sink to the bottom of the petri dish.
 +
<br/><br/>
 +
4. Add 30ul of the prepared sample into the center of the paraffin layer. Measure and record the diameter of the water circle the expands from the center. Do this immediately, otherwise the circle will shrink and lead to systematic error.
 +
  <h1 id="putida">Mutant rhlC <em>P. putida</em> produces
 +
    di-rhamnolipids</h1>
 +
 
 +
  <h2>Quantification of rhamnolipids</h2>
 +
  <p>
 +
    In order to accurately measure the amount of
 +
    rhamnolipids produced by our mutant strains, we
 +
    used supercritical fluid chromatography
 +
    (SFC-MS). First, a test run was executed with a
 +
    mixture of mono-rhamnolipids and di-rhamnolipids
 +
    at the concentration of 5 mg/mL by running the
 +
    sample through the column packed with 2-PIC. From
 +
    this test run, we have obtained the retention
 +
    times of mono-rhamnolipids (rha-C<sub>10</sub>-C<sub>10</sub>:
 +
    pseudomolecular ion of 503.56 m/z) and
 +
    di-rhamnolipids (rha-rha-C<sub>10</sub>-C<sub>10</sub>: pseudomolecular
 +
    ion of 649.8 m/z) to be approximately 3.974 min
 +
    and 4.942 min respectively.  The mass
 +
    fractions were obtained from electrospray
 +
    ionization (ESI) negative mode.
 +
  </p>
 +
  <p>
 +
    From our TLC analysis, it was found that
 +
    supplementing the LB media with glucose is crucial
 +
    to the production of rhamnolipid. Therefore, for
 +
    SFC-MS analysis, our mutant strain <em>P. putida</em> RhlABC was grown in LB supplemented
 +
    with 50 g/L of glucose for 24 hours.  From the SFC-MS data, it was found
 +
    that approximately 142 &#x00b5;g/mL of
 +
    rha-C<sub>10</sub>-C<sub>10</sub> and 3.524 &#x00b5;g/mL of
 +
    rha-rha-C<sub>10</sub>-C<sub>10</sub> were detected.
 +
  </p>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/parts/9/9c/RhlABC1.png" alt="mono-rhamnolipids production" width="700">
 +
  </figure>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/parts/a/ab/RhlABC2.png" alt="di-rhamnolipids production" width="700">
 +
  </figure>
 +
 +
<h1 id="Mosqitoes">Di-Rhamnolipids repel <em>Aedes Aegypti</em></h1>
 +
<p>
 +
In order to quantify how effectively rhamnolipids repel mosquitoes, we conducted mosquito feeding and landing assays. <em>Aedes aegypti</em>, the species of mosquito observed to carry Zika virus, were grown from larval stage, and females were sorted at the pupae or adult stage. Since only females consume blood for reproduction, we were only interested in using them for the assays.
 +
</p>
 
<p>
 
<p>
−
                    In order to quantify how effectively rhamnolipids
+
One day before experiment, 50 total mosquitos (with 30 females) were isolated in cages and starved from 23-25 hours. Each cage was then taken to a warm room (~30 oC), and the cage was covered with wet paper towels to preserve humidity.  For each trial, our blood feeding system (Figure) was placed on top of the cage each with a cotton gauze soaked with either negative control water, 1 mg/mL mono-rhamnolipid solution, 1 mg/mL di-rhamnolipid solution, or positive control 25% DEET, and the mosquito activity was videotaped for 1 hour.  Afterwards, the cage was taken to the cold room to paralyze the assayed mosquitoes, and mosquitoes that had consumed blood were counted.  It is important to note that the age of female mosquitoes and the time of feeding played an important role in how mosquitoes behave. Typically, it is optimum to use female mosquitoes of age from 4-6 days for feeding assays as any mosquitoes older than this age range will be too old to reproduce, and thereby not needing to drink blood.  Furthermore, their feeding is most active 4 hours before dusk.  Some of our trials that didn’t meet these criteria did not result in any feeding, but we did observe significant difference in landing between the control and rhamnolipids. Our landing assay results showed that while DEET was the strongest mosquito repellent with no landings or fed mosquitos, 1 mg/mL mono and di-rhamnolipid still showed statistically significant repulsion as shown in the graph below.
−
                    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>
 
</p>
 +
<figure>
 +
    <img src="https://static.igem.org/mediawiki/parts/8/8a/Mosquito.png"
 +
alt="Mosquito Experiment" width="650">
 +
</figure>
 +
<h1 id="keratinocytes"><em>P. putida</em>, <em>S. epidermidis</em>,
 +
    and rhamnolipids are compatible with human
 +
    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;g/mL and 65.52 &#x00b5;g/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</em> PAK, <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">3</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>
 +
    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 <em>S. aureus</em> strain (RN4220) and our
 +
    <em>S. epidermidis</em> (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 <em>S. epidermidis</em> 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>
 +
    <figure>
 +
    <img src="https://static.igem.org/mediawiki/parts/3/30/Staph_rhamno.png"
 +
alt="S. Epidermidis Growth in the presence of rhamnolipids" width="600">
 +
  </figure>
  
  
 +
  <hr>
 +
<p>
 +
<sup><a href="http://doi.org/10.1007/978-3-642-14490-5_2">1</a></sup>
 +
Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.<br>
 +
<sup><a href="http://doi.org/10.3389/fmicb.2015.00088">2</a></sup>
 +
Silva, Vinicius L., Roberta B. Lovaglio, Claudio J. Zuben, and Jonas Contiero. "Rhamnolipids: Solution against Aedes Aegypti?" Frontiers. Frontiers in Microbiology, 16 Feb. 2015. Web. 23 Oct. 2016.<br>
 +
Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.<br>
 +
    <sup><a href="http://www.biovision.com/manuals/K300.pdf">3</a></sup>
 +
    "MTS Cell Proliferation Colorimetric Assay Kit."
 +
    BioVision. Web.
 +
  </p>
 +
</html>
  
−
<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>
 
  
  

Latest revision as of 05:34, 21 October 2021


rhamnosyltransferase 2 [Pseudomonas aeruginosa]

Rhamnolipids, a class of glycolipids characterized by a rhamnose moiety attached to a fatty acid tail, is produced by many organisms—with the Pseudomonas aeruginosa as the most predominate. We have shown that Pseudomonas putida produces both mono-rhamnolipids and di-rhamnolipids with the addition of the rhlAB and rhlC operons, respectively. Previous research has shown that di-rhamnolipids repel the Aedes aegypti mosquito. We have shown that both di-rhamnolipids and mono-rhamnolipids repel Aedes aegypti. We have also shown that rhamnolipids are compatible with human keratinocytes in the presence of both Pseudomonas aeruginosa and Pseudomonas putida. Lastly, we have shown that rhamnolipids are compatible with Staphylococcus epidermidis—a skin microbiome organism.

Introduction

Rhamnolipids are a class of glycolipids characterized by a rhamnose moiety and a fatty acid tail. While rhamnolipids are produced in a variety of organisms, Pseudomonas aeruginosa is most frequently cited. In Pseudomonas aeruginosa, genes rhlA and rhlB are cooperative to from the complex rhlAB that codes for the enzyme rhamnosyltransferase 1. The enzyme rhamnosyltransferase 1 catalyzes the addition of a (hydroxyalkanoyloxy)alkanoic acid (HAA) fatty acid tail to a rhamnose sugar to produce a mono-rhamnolipid. Similarly, rhlC codes for the enzyme rhamnosyltransferase 2, which catalyzes an addition of another rhamnose moiety to a mono-rhamnolipid to form a di-rhamnolipid.

Rhamnolipids are predominantly known for their biosurfactant properties, which possesses industrial applications 1. Di-rhamnolipids have also been shown to repel the Aedes aegypti mosquito 2. In our investigation, we have confirmed with statistical significance that di-rhamnolipids repel Aedes aegypti. We have also shown with statistical significance that mono-rhamnolipids repel Aedes aegypti. The compatibility of rhamnolipids with human skin was also a main concern of ours—as rhamnolipids have been shown to be a virulence factor. We have shown that rhamnolipids are compatible with human keratinocytes in the presence of both Pseudomonas aeruginosa and Pseudomonas putida. Likewise, we have shown that rhamnolipids are compatible with Staphylococcus epidermidis—a skin microbiome organism. Lastly, we have confirmed the both mono-rhamnolipids and di-rhamnolipids are producible in Pseudomonas putida with the addition of rhlAB and rhlC, respectively.

Convenient quantification of rhamnolipid（BNDS China 2021）.

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

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

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

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

Method:

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

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

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

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

Mutant rhlC P. putida produces di-rhamnolipids

Quantification of rhamnolipids

In order to accurately measure the amount of rhamnolipids produced by our mutant strains, we used supercritical fluid chromatography (SFC-MS). First, a test run was executed with a mixture of mono-rhamnolipids and di-rhamnolipids at the concentration of 5 mg/mL by running the sample through the column packed with 2-PIC. From this test run, we have obtained the retention times of mono-rhamnolipids (rha-C10-C10: pseudomolecular ion of 503.56 m/z) and di-rhamnolipids (rha-rha-C10-C10: pseudomolecular ion of 649.8 m/z) to be approximately 3.974 min and 4.942 min respectively. The mass fractions were obtained from electrospray ionization (ESI) negative mode.

From our TLC analysis, it was found that supplementing the LB media with glucose is crucial to the production of rhamnolipid. Therefore, for SFC-MS analysis, our mutant strain P. putida RhlABC was grown in LB supplemented with 50 g/L of glucose for 24 hours. From the SFC-MS data, it was found that approximately 142 µg/mL of rha-C10-C10 and 3.524 µg/mL of rha-rha-C10-C10 were detected.

mono-rhamnolipids production
di-rhamnolipids production

Di-Rhamnolipids repel Aedes Aegypti

In order to quantify how effectively rhamnolipids repel mosquitoes, we conducted mosquito feeding and landing assays. Aedes aegypti, the species of mosquito observed to carry Zika virus, were grown from larval stage, and females were sorted at the pupae or adult stage. Since only females consume blood for reproduction, we were only interested in using them for the assays.

One day before experiment, 50 total mosquitos (with 30 females) were isolated in cages and starved from 23-25 hours. Each cage was then taken to a warm room (~30 oC), and the cage was covered with wet paper towels to preserve humidity. For each trial, our blood feeding system (Figure) was placed on top of the cage each with a cotton gauze soaked with either negative control water, 1 mg/mL mono-rhamnolipid solution, 1 mg/mL di-rhamnolipid solution, or positive control 25% DEET, and the mosquito activity was videotaped for 1 hour. Afterwards, the cage was taken to the cold room to paralyze the assayed mosquitoes, and mosquitoes that had consumed blood were counted. It is important to note that the age of female mosquitoes and the time of feeding played an important role in how mosquitoes behave. Typically, it is optimum to use female mosquitoes of age from 4-6 days for feeding assays as any mosquitoes older than this age range will be too old to reproduce, and thereby not needing to drink blood. Furthermore, their feeding is most active 4 hours before dusk. Some of our trials that didn’t meet these criteria did not result in any feeding, but we did observe significant difference in landing between the control and rhamnolipids. Our landing assay results showed that while DEET was the strongest mosquito repellent with no landings or fed mosquitos, 1 mg/mL mono and di-rhamnolipid still showed statistically significant repulsion as shown in the graph below.

Mosquito Experiment

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 µg/mL and 65.52 µg/mL. Relating the results to rhamnolipid quantification, the concentration of rhamnolipid the construct produces should not cause significant cell death.

Keratinocyte cell viability bacteria assay

Keratinocyte species

Keratinocytes were co-cultured with different strains of bacteria (Pseudomonas putida, Pseudomonas aeruginosa PAK, Staphylococcus aureus, Staphylococcus epidermidis, and mutant rhlAB P. putida). Half were cultured in plain DMEM with serum, and half were culture in DMEM with 1 mg/mL mixed mono- and di- rhamnolipids. After co-culturing, the keratinocytes were washed with PBS, exposed to gentamicin in an attempt to kill the bacteria, and incubated in MTS cell viability assay for up to 4 hours and viewed in a plate reader. MTS assay is colorimetric cell viability assay and reacts with NADPH-dependent dehydrogenase enzymes, which are only active in live (metabolically active) cells3. For the MTS assay, pure media were used as a negative control (100% cell death), and keratinocyte culture with normal DMEM was used as a positive control (“0%” cell death, or the maximum number of cells that could be alive).

Keratinocyte P. putida coculture

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.

S. Epidermidis Growth in the presence of rhamnolipids

1 Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.
2 Silva, Vinicius L., Roberta B. Lovaglio, Claudio J. Zuben, and Jonas Contiero. "Rhamnolipids: Solution against Aedes Aegypti?" Frontiers. Frontiers in Microbiology, 16 Feb. 2015. Web. 23 Oct. 2016.
Abdel-Mawgoud, Ahmad M., Rudolf Hausmann, Francois Lepine, Markus M. Muller, and Eric Deziel. "Rhamnolipids: Detection, Analysis, Biosynthesis, Genetic Regulation, and Bioengineering of Production." Springer Link. Microbiology Monographs, 14 Sept. 2010. Web. 20 Oct. 2016.
3 "MTS Cell Proliferation Colorimetric Assay Kit." BioVision. Web.


Sequence and Features


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