Difference between revisions of "Part:BBa K424018"

 
 
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<partinfo>BBa_K424018 short</partinfo>
 
<partinfo>BBa_K424018 short</partinfo>
  
Pseudomonas aeruginosa species of bacteria produce the rhamnosyltransferase 1 complex (RhlAB) that is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid.
+
''Pseudomonas aeruginosa'' species of bacteria produce the natural rhamnosyltransferase gene complex (RhlAB) that is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid that is a biomolecule with biosurfactant propierties but the natural RhlAB gene have illegal restriction sites that make it incompatible with the Assembly Standard 10.
How to used it?
+
 
We developed this standarized part to be inserted into E. coli for rhamnolipid production through the key enzyme rhamnosyltranferase. For this we provide to E. coli the proper sustrate to rhamnosiltransferase biosinthesize rhamnolipids in vitro.
+
How to used it as a BioBrick?
 +
We developed this standarized part to be inserted into ''E. coli'' for rhamnolipid production through the mutation of the key enzyme rhamnosyltranferase (RhlAB) to be compatible with Assembly Standard 10 . For this we do to the natural RhlAB gene a QuikChange Lightning Multi Site-Directed Mutagenesis following the Kit manual Instruction  from Stratagene. This rhamnosyltransferase BioBrick (Rh1AB_BB) is ready to be tested on a test plataform device according to the Assembly Standard Protocol 10 from parts of the registry.
 +
 
 +
== '''Further Characterization by ColumbiaU_NYC iGEM 2016 team''' ==
 +
 
 +
<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> mosquitoes. 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 form 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>
 +
 
 +
  <h1 id="putida">Mutant rhlAB <em>P. putida</em> produces
 +
    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. Then, a calibration
 +
    curve was constructed with 95% pure
 +
    mono-rhamnolipids, and the limit of detection was
 +
    found to be approximately 5 &#x00b5;g/mL. The mass
 +
    fractions were obtained from electrospray
 +
    ionization (ESI) negative mode.
 +
  </p>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/parts/c/c9/Sfc-calibration.png" alt="Calibration" width="600">
 +
  </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
 +
    (<em>E. coli</em>_H2_RhlAB, <em>E. coli</em>_L1_RhlAB, and
 +
    <em>P. putida</em>_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>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/2016/a/a3/Ecoli-sfc-2.png" alt="E. coli" width="700">
 +
  </figure>
 +
  <figure>
 +
    <img src="https://static.igem.org/mediawiki/2016/2/29/Pputida-sfc.png" alt="P. putida" 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>
 +
<h1 id="Mosqitoes">Mono-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>
 +
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.
 +
</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>
 +
 
  
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  

Latest revision as of 05:30, 28 October 2016

Rhamnosiltransferase BioBrick (Rh1AB_BB)

Pseudomonas aeruginosa species of bacteria produce the natural rhamnosyltransferase gene complex (RhlAB) that is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid that is a biomolecule with biosurfactant propierties but the natural RhlAB gene have illegal restriction sites that make it incompatible with the Assembly Standard 10.

How to used it as a BioBrick? We developed this standarized part to be inserted into E. coli for rhamnolipid production through the mutation of the key enzyme rhamnosyltranferase (RhlAB) to be compatible with Assembly Standard 10 . For this we do to the natural RhlAB gene a QuikChange Lightning Multi Site-Directed Mutagenesis following the Kit manual Instruction from Stratagene. This rhamnosyltransferase BioBrick (Rh1AB_BB) is ready to be tested on a test plataform device according to the Assembly Standard Protocol 10 from parts of the registry.

Further Characterization by ColumbiaU_NYC iGEM 2016 team

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 mosquitoes. 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 form the complex rhlAB that codes for the enzyme rhamnosyltransferase 1. The enzyme rhamnosyltransferase 1 catalyzes the addition of a (hydroxyalkanoyloxy) alkanoic acid (HAA) fatty acid tail to a rhamnose sugar to produce a mono-rhamnolipid. Similarly, rhlC codes for the enzyme rhamnosyltransferase 2, which catalyzes an addition of another rhamnose moiety to a mono-rhamnolipid to form a di-rhamnolipid.

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

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

Calibration

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_H2_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
E. coli
P. putida

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.

Mono-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.


Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 169
    Illegal BamHI site found at 729
    Illegal XhoI site found at 905
    Illegal XhoI site found at 2191
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1084
    Illegal NgoMIV site found at 1805
    Illegal NgoMIV site found at 1918
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 394
    Illegal BsaI site found at 1434
    Illegal BsaI.rc site found at 578