Team:Paris Bettencourt/Notebook/Old People Smell

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<body>
<div id="topheader">
<div id="topheader">
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<img id="PBlogo" src="https://static.igem.org/mediawiki/2013/3/3a/PB_logoParis.gif"/>
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<img src="https://static.igem.org/mediawiki/2014/6/6f/Teenspirit.png" class=nameimg>
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<a href="https://2014.igem.org/Main_Page"> <img id="iGEMlogo" src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a>
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<h1></h1>
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<h2>Paris Bettencourt 2014</h2>
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</div>
</div>
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<div id=moocdiv>
-
<table id=menu>
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<table>
-
<tr>
+
<tr>
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<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt" id = "link">Home </a> </td>
+
<td><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Odor_Library">Smell The Roses</a></td>
-
+
<td><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/TMAU">Something Fishy</a></td>
-
<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt/Team" id = "link"> Team </a> </td>
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<td><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Eliminate_Smell">Don't Sweat It</a></td>
-
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<td><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Foot_Odor">Goody Two Shoes</a></td>
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<td id = "cadre"><a href="https://igem.org/Team.cgi?year=2014&team_name=Paris_Bettencourt" id = "link"> Official Team Profile </a></td>
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<td><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell">Teen Spirit</a></td>
-
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<td><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Interlab_Study">Interlab Study</a></td>
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<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt/Project" id = "link"> Project</a></td>
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</tr>
-
+
</table>
-
<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt/Parts" id = "link"> Parts</a></td>
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</div>
-
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                <div id=intra>
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<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt/Modeling" id = "link"> Modeling</a></td>
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-
+
-
<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook" id = "link"> Notebook</a></td>
+
-
+
-
<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt/Safety" id = "link"> Safety </a></td>
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-
+
-
<td id = "cadre"><a href="https://2014.igem.org/Team:Paris_Bettencourt/Attributions" id = "link"> Attributions </a></td>
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-
</tr>
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-
</table>
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-
</head>
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-
+
-
<body>
+
-
+
-
</br>
+
-
</br>
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-
+
-
<div id="shadow">
+
-
<ul id="menu-accordeon">
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-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Odor_Library" id="link">+ Odor Library</a>
+
-
<ul>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Odor_Library#June">June</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Odor_Library#July">July</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Odor_Library#August">August</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Odor_Library#September">September</a></li>
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-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Odor_Library#October">October</a></li>
+
-
</ul>
+
-
</li>
+
-
<li>+ Probiotics and Cosmetics
+
-
<ul>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Probiotics#June">June</a></li>
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-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Probiotics#July">July</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Probiotics#August">August</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Probiotics#September">September</a></li>
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-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Probiotics#October">October</a></li>
+
-
</ul>
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-
</li>
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-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell" id="link">+ Old People Smell</a>
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-
<ul>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#June">June</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#July">July</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#August">August</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#September">September</a></li>
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-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#October">October</a></li>
+
-
</ul>
+
-
</li>
+
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/TMAU" id="link">+ TMAU</a>
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<ul>
<ul>
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/TMAU#June">June</a></li>
+
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#June">June</br></br></a></li>
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/TMAU#July">July</a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#July">July</br></br></a></li>
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/TMAU#August">August</a></li>
+
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#August">August</br></br></a></li>
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/TMAU#September">September</a></li>
+
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#September">September</br></br></a></li>
-
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/TMAU#October">October</a></li>
+
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Notebook/Old_People_Smell#October">October</br></br></a></li>
</ul>
</ul>
-
</li>
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                </div>
-
</ul>
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                </br>
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</div>
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                </br>
 +
 
<div id="corps">
<div id="corps">
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<h3>Notebook</h3>
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                <p id=top><a href="#haut"><img src="https://static.igem.org/mediawiki/2014/4/41/Arrow.png"></a></p>
-
                        <h3>Old Person odor</h3>
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<h3 id=haut>Notebook</h3>
 +
 
 +
              <!-- Template <h5>Date 1</h5>
 +
                                        <h6>Goal</h6>
 +
<p>Text</p>
 +
                                        <h6>Procedure</h6>
 +
<p>Text</p>
 +
                                        <h6>Results</h6>
 +
<h5>Date 2</h5>
 +
                                        <h6>Goal</h6>
 +
<p>Text</p>
 +
                                        <h6>Procedure</h6>
 +
<p>Text</p>
 +
                                        <h6>Results</h6>  -->
 +
 
<div id="August">
<div id="August">
 +
                                </br>
<h4>August</h4>
<h4>August</h4>
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<h5 id="date">August 5th 1</h5>
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                                        </br>
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<p id="text">The goal is to select a bacteria able to develop on 2-nonenal.
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<h5 id="date">August 5th </h5>
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We made 2 new media:</p>
+
<p id="text">The goal is to select a bacteria able to develop on 2-nonenal. We made 3 media:</p>
 +
<p id="text"><u> 1) M9 Minimal Media 100% Glucose</u></p>
 +
                                        <ul>
 +
                                    <li>200mL M9 Salts</li>
 +
                                    <li>0.1mL CaCl2 1M</li>
 +
                                            <li>2mL MgSO4 1M</li>
 +
                                            <li>20mL Glucose 20%</li>
 +
                                            <li> Water: bring to 1L</li>
 +
                                        </ul>
 +
                                        </br>
 +
<p id="text"><u> 2) M9 Minimal Media 50% Glucose - 50% 2-Nonenal (according to the number of carbon atoms)</u></p>
 +
                                        <ul>
 +
                                    <li>200mL M9 Salts</li>
 +
                                    <li>0.1mL CaCl2 1M</li>
 +
                                            <li>2mL MgSO4 1M</li>
 +
                                            <li>10mL Glucose 20%</li>
 +
                                            <li>10mL 2-nonenal 10,7%*</li>
 +
                                            <li> Water: bring to 1L</li>
 +
                                        </ul>
 +
 
 +
                                        </br>
 +
<p id="m9non"><u> 3) M9 Minimal Media 100% 2-nonenal</u></p>
 +
                                        <ul>
 +
                                    <li>200mL M9 Salts</li>
 +
                                    <li>0.1mL CaCl2 1M</li>
 +
                                            <li>2mL MgSO4 1M</li>
 +
                                            <li>20mL 2-nonenal 10,7%*</li>
 +
                                            <li> Water: bring to 1L</li>
 +
                                        </ul>
 +
                                        </br>
 +
                                        <p>We autoclaved 6 bottles (2 of 500mL for each)</p>
 +
                                        <p>Then, we made liquid culture of C.striatum in media <b>1)</b> to try if the minima media works.</p>
</br>
</br>
-
<p id="text"><b>1) M9 Minimal Media 100% Glucose</b></p>
+
                                        <p><i>* Concentration of the 2-nonenal solution:
-
<h5 id="date">Date 2</h5>
+
In M9 Media, we use 20mL of Glucose 20% (200g/L). The concentration of glucose is C=200/M=1.09 mol/L.
-
<p id="text">Text</p>
+
Glucose has 6 carbons while 2-nonenal has 9, so we need 2/3 of the glucose volume to have the same amount of carbon atoms. C=2/3*1.09=0.74 mol/L and then 0.74*M=103.7g/L. That's why the concentration of the 2-nonenal solution is 10.73%. We also added Tween80.</i></p>
-
<p id="text">Text</p>
+
                                        </br>
-
</br>
+
                                        </br>
 +
  <h5 id="date">August 6th</h5>
 +
<p id="text">We poured 20 plates of each of the 3 media with 350mL of an agar solution and 100mL of medium.</p>
 +
<p id="text">Then we used 3 plates of each type to culture skin bacteria from 3 spots: arm, armpit and forehead. Ursczula sampled bacteria from her skin with a loop and for each spot she diluted the bacteria in 300uL of NF Water. Then we plated 100uL of each solution in each of the 3 types of plates and incubated them at 37°C.</p>
 +
                                        <TABLE BORDER>
 +
<TR><TD></TD><TD><b>M9 100% glucose</b></TD><TD><b>M9 50%-50%</b></TD><TD><b>M9 100% 2-nonenal</b></TD></TR>
 +
<TR><TD><b>Arm</b></TD></TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
<TR><TD><b>Armpit</b></TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
<TR><TD><b>Forehead</b></TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
<TR><TD><b>NEB (control)</b></TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
</TABLE>
 +
        </br>
 +
                                        </br>
 +
                                        <h5 id="date">August 8th </h5>
 +
                                        <p id="text">After 2 days, nothind has developped on the M9 plates. M9 media is actually really slow for the development of any strain. That is why we created a new media which would be like the real skin environment: synthetic sweat according to <a href="https://static.igem.org/mediawiki/2014/1/15/Artificial_sweat.pdf">this recipe</a></p>
 +
                                        <p>According to the recipe, fatty acids seem to be the main source of carbon in synthetic sweat so we replaced them by 2-nonenal.</br>
 +
We made 2 media:</br>
 +
1) without fatty acids </br>
 +
2) without fatty acids and with 2-nonenal mixed with 10% of Tween 80.
 +
 
 +
                                        </br>
 +
                                        </br>
 +
                                        <h5 id="date">August 11th</h5>
 +
                                        <p id="text"><u> Results of M9 plates after 5 days</u></p>
 +
                                        <TABLE BORDER>
 +
<TR><TD></TD><TD><b>M9 100% glucose</b></TD><TD><b>M9 50%-50%</b></TD><TD><b>M9 100% 2-nonenal</b></TD></TR>
 +
<TR><TD><b>Arm</b></TD><TD>Many very small colonies</TD><TD>6</TD><TD>0</TD></TR>
 +
<TR><TD><b>Armpit</b></TD><TD>Many very small colonies</TD><TD>0</TD><TD>0</TD></TR>
 +
<TR><TD><b>Forehead</b></TD><TD>27</TD><TD>5</TD><TD>0</TD></TR>
 +
<TR><TD><b>NEB (control)</b></TD><TD>Many</TD><TD>many</TD><TD>0</TD></TR>
 +
</TABLE>
 +
<p>Analysis: </br>
 +
The fitness of bacteria is better on 100% Glucose than on 50-50%. No colony develops on 2-nonenal, not even NEB. Then we can suggest that 2-nonenal is not used as a carbon source and that the difference of fitness between 100% glucose and 50-50% is only due to the lower amount of glucose in 50-50%. We should have made plates with only 50% of glucose to compare. </p>
 +
</br>
 +
<p> We also plated samples on synthetic sweat: </p>
 +
                                        <TABLE BORDER>
 +
                                        <TR><b><TD></TD><TD>NEB</TD><TD>Forehead</TD><TD>Nose</TD></b></TR>
 +
                                        <TR><TD><b>1) Without fatty acids</b></TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
                                        <TR><TD><b>2) Without fatty acids and with 2-nonenal</b></TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
                                        </TABLE>
 +
                                        </br>
 +
                                        <p> On August 10th, Jake tried a new medium with peptone added to M9. He made 2 media (20 plates of each):
 +
                                        <ul>
 +
                                            <li><u>iGEM medium 1 (100% glucose)</u></li>
 +
                                            <ul>
 +
                                                  <li>pancreatic digest of casein: 15 g</li>
 +
                                                  <li>glucose: 5 g</li>
 +
                                                  <li>NaCl: l5 g</li>
 +
                                                  <li>Agar: 15 g</li>
 +
                                                  <li>Water: 1 L</li>
 +
                                            </ul>
 +
                                            <li><u> iGEM medium 2 (100% 2-nonenal):</u></li>
 +
                                            <ul>
 +
                                                  <li>pancreatic digest of casein: 15 g</li>
 +
                                                  <li>2-nonenal: 10 g</li>
 +
                                                  <li>Tween 80: 250uL (premix with 2-nonenal before adding to the medium)</li>
 +
                                                  <li>NaCl: l5 g</li>
 +
                                                  <li>Agar: 15 g</li>
 +
                                                  <li>Water: 1 L</li>
 +
                                            </ul>
 +
                                        </ul>
 +
                                        </br><p>I prepared 2 new media:</p>
 +
                                        <ul>
 +
                                            <li><u>iGEM medium 3 (50% glucose)</u></li>
 +
                                            <ul>
 +
                                                  <li>pancreatic digest of casein: 7.5 g</li>
 +
                                                  <li>glucose: 1.25 g</li>
 +
                                                  <li>NaCl: 7.5 g</li>
 +
                                                  <li>Agar: 7.5 g</li>
 +
                                                  <li>Water: 0.5 L</li>
 +
                                            </ul>
 +
                                            <li><u> iGEM medium 4 (50% glucose - 50% 2-nonenal):</u></li>
 +
                                            <ul>
 +
                                                  <li>pancreatic digest of casein: 7.5 g</li>
 +
                                                  <li>glucose: 1.25 g</li>
 +
                                                  <li>2-nonenal: 2.5 g</li>
 +
                                                  <li>Tween 80: 125uL (premix with 2-nonenal before adding to the autoclaved medium)</li>
 +
                                                  <li>NaCl: 7.5 g</li>
 +
                                                  <li>Agar: 7.5 g</li>
 +
                                                  <li>Water: 0.5 L</li>
 +
                                            </ul>
 +
                                        </ul>
 +
                                        </br>
 +
                                        <p> I autoclaved them at 111°C - 30min (programm 5). </p>
 +
<p> Then, I added the 2-nonenal to iGEM medium 4: density of nonenal is 0.846g/mL so I put 2.5/0.846 = 2.96 mL of 2-nonenal mixed with 125uL of Tween 80. I poured 20 plates of each medium and then plated before incubating at 37°C:</p>
 +
 
 +
<TABLE BORDER>
 +
<TR><b><TD></TD><TD>iGEM medium 1</TD><TD>iGEM medium 2</TD><TD>iGEM medium 3</b></TD><TD>iGEM medium 4</TD></b></TR>
 +
<TR><TD><b>Nose</b></TD><TD>x</TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
<TR><TD><b>Forehead</b></TD><TD>x</TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
<TR><TD><b>NEB (control)</b></TD><TD>x</TD><TD>x</TD><TD>x</TD><TD>x</TD></TR>
 +
</TABLE>
 +
                                        </br>
 +
                                        </br>
 +
                                        <h5 id="date">August 13th</h5>
 +
<p id="text"><u>Results of M9 plates after 7 days</u></p>
 +
                                        <TABLE BORDER>
 +
<TR><TD></TD><TD><b>M9 100% glucose</b></TD><TD><b>M9 50%-50%</b></TD><TD><b>M9 100% 2-nonenal</b></TD></TR>
 +
<TR><TD><b>Arm</b></TD><TD>Many very small colonies</TD><TD>17 including 6 big ones</TD><TD>6 very small?</TD></TR>
 +
<TR><TD><b>Armpit</b></TD><TD>Many very small colonies</TD><TD>About 20 very small</TD><TD>1 or 2 really small?</TD></TR>
 +
<TR><TD><b>Forehead</b></TD><TD>32</TD><TD>8</TD><TD>1</TD></TR>
 +
<TR><TD><b>NEB (control)</b></TD><TD>Many</TD><TD>many</TD><TD>0</TD></TR>
 +
</TABLE>
 +
 
 +
<p> I plated on LBA the colony developped on 100% 2-nonenal from the Urszula's forehead to developp it.</p>
 +
                                        </br>
 +
                                        </br>
 +
                                        <h5 id="date">August 14th</h5>
 +
<p id="text">The colony developped on M9 100% 2-nonenal developped perfectly on LBA plates. It seems to be Staphylococcus aureus. I transfered colonies on 3 M9 100% 2-nonenal plates and I started 2 liquid cultures: one in LB and one in M9 100% 2-nonenal.</p>
 +
</br>
 +
                                        </br>
 +
                                        <h5 id="date">August 17th</h5>
 +
<p id="text">We started new approach: Directed evolution of forehead, armpit and nose skin samples  in the liquid medium </br>
 +
First two forehead samples  in 37°C in medium 95% (4.75mL) LB and 5% M9 Minimal Media 100% 2-nonenal (0.25 mL)
 +
</p>
 +
                                        </br>
 +
                                        </br>
 +
                                        <h5 id="date">August 18th</h5>
 +
<p id="text"><u>1) After four days of culture, I measured the absorbance of the liquid cultures of Staphylococcus aureus in LB and in M9 100% 2-nonenal medium.</u></p>
 +
                                        <TABLE BORDER>
 +
<TR><TD></TD><TD><b>A</b></TD></TR>
 +
<TR><TD><b>LB</b></TD><TD>1.647</TD></TR>
 +
<TR><TD><b>M9 100% 2-nonenal medium</b></TD><TD>0.109</TD></TR>
 +
</TABLE>
 +
 
 +
<p> S.aureus still develops on 2-nonenal but its fitness is much lower than on LB. I let it grow a few days more.</p>
 +
</br>
 +
<p id="text"><u>2) M9 plates from August 6th</u></p>
 +
<p> It seems like we got small colonies on every 100% 2-nonenal plate (NEB, Forehead, Arm and Armpit). For each one, I prepared 2 liquid cultures: 1 LB and 1 M9 100% 2-nonenal.</p>
 +
</br>
 +
<p id="text"><u>3)Directed evolution</u></p>
 +
<p> Absorbance has been measured for 2 tubes. A = 0 in both tubes. I will give it one more day</p>
 +
 
 +
</br>
 +
 
 +
<p> For an armpit and nose skin samples I proceeded similarly as started yesterday: incubated in 37°C in medium 95% (4.75mL) LB and 5% <a href="#m9non">M9 Minimal Media</a> 100% 2-nonenal (0.25 mL) with a sample from my armpit and another sample from my nose skin.</p>
</div>
</div>
 +
</br>
 +
                                        </br>
 +
                                        <h5 id="date">August 19th</h5>
 +
<p id="text"><u>1) Directed evolution - gradual approach</u></p>
 +
<p>Samples 1 and 2 in 95%LB:5% M9 2-nonenal medium grew well and the new armpit and nose skin samples as well</p>
 +
                                        <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<!--<TR><TD>19.08 12h35</TD><TD>2.31</TD><TD>1.54</TD><TD>90% LB:10% 2-nonenal</TD></TR>-->
 +
</TABLE><p id="text">I centrifuged all 4 samples, discarded supernatant and changed the medium to 90% LB and 10% M9 2-nonenal</p>
 +
</br>
 +
                                        <p><u> 2) Staphylococcus aureus </u></p>
 +
<p> I plated on LBA the forehead colonies from August 15th which had developped on 2-nonenal. Next week we will make a olfactive test to estimate the decrease of 2-nonenal using S.aureus. This strain will be called (N+)-S.aureus.
 +
</br>
 +
                                        </br>
 +
                                        <h5 id="date">August 20th</h5>
 +
<p id="text"><u>1) Directed evolution - gradual approach</u></p>
 +
<p><i> Corynebacterium striatum</i> and nose and forehead samples density in 90%LB:10% M9 2-nonenal medium  </p>
 +
                                        <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
</TABLE><p id="text">I centrifuged all 4 samples, discharged supernatant and changed the medium to 85% LB and 15% M9 2-nonenal</p>
 +
</br>
 +
<p><u> 2) Plates</u> (Samples 1 and 2)</p>
 +
The LB plate of (N+)-S.aureus has many colonies. I made a liquid culture to get a glycerol stock.
 +
 +
<p><u> 3) New M9</u></p>
 +
<p>I prepared 2x50mL of each media:</p>
 +
<ul><li>M9 - 2-nonenal (120uL mixed for 3uL of Tween80 added after autoclave for 50mL of medium)</li>
 +
    <li>M9 - 2-nonenal (120uL mixed for 3uL of Tween80 added after autoclave for 50mL of medium) + 0.5g of peptone for 50mL of medium</li>
 +
</ul>
 +
<p> I want to try to make M9 a faster medium. I started a liquid culture of (N+)-S.aureus and measured the initial density of each medium.</p>
 +
<TABLE BORDER>
 +
<TR><TD></TD><TD><b>OD600</b></TD></TR>
 +
<TR><TD><b>M9 - 2-nonenal</b></TD></TD><TD>0.885</TD></TR>
 +
<TR><TD><b>M9 - 2-nonenal + peptone</b></TD><TD>0.994</TD></TR>
 +
</TABLE>
 +
</br>
 +
                                        </br>
 +
                                        <h5 id="date">August 21th</h5>
 +
<p><u>1) Glycerol stock</u></p>
 +
<p> I made a glycerol stock using (N+)-S.aureus liquid culture in LB according to <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot8" > this protocol</a>. The strain is now called sPB.050
 +
</br>
 +
<p><u>2) M9 overnight liquid cultures</u></p>
 +
<p> After 24 hours, I measured the OD600 of the M9 liquid cultures with or without 2-nonenal.
 +
<TABLE BORDER>
 +
<TR><TD></TD><TD><b>OD600 before culture</b></TD><TD><b>OD600 after 24H culture</b></TD></TR>
 +
<TR><TD><b>M9 - 2-nonenal</b></TD><TD>0.885</TD><TD>0.424</TD></TR>
 +
<TR><TD><b>M9 - 2-nonenal + peptone</b></TD><TD>0.994</TD><TD>0.511</TD></TR>
 +
</TABLE>
 +
<p>The result can be surprising because OD600 has decreased. But we figured out thant the main element which sets OD600 is the concentration of 2-nonenal because our medium is an emulsion. That is why OD600 is really variable according to the mixture. There is here a balance between the bacterian growth which increases OD600, correlated to a decrease of 2-nonenal concentration which lowers OD600 because it is consumed by the cells. It would be interesting to plot OD600 as a function of 2-nonenal concentration and CFU.</p>
 +
<p> To plot OD600 as a function of CFU, I used the (N+)-S.aureus liquid culture in LB, putting different volumes in each tube. Then I centrifuged the tubes (1min-5000tr/min), discarded the supernatant, added 1mL of PBS and resuspended the cell by vortexing. I measured OD600 (with 500uL of solution) and then plated the solutions (100uL in each plate).
 +
<TABLE BORDER>
 +
<TR><TD><b>Tube</b></TD><TD><b>Volume of LB culture (uL)</b></TD><TD><b>OD600</b></TD></TR>
 +
<TR><TD>1</TD><TD>1000</TD><TD>2.581</TD></TR>
 +
<TR><TD>2</TD><TD>500</TD><TD>1.758</TD></TR>
 +
<TR><TD>3</TD><TD>250</TD><TD>1.104</TD></TR>
 +
<TR><TD>4</TD><TD>200</TD><TD>0.927</TD></TR>
 +
<TR><TD>5</TD><TD>150</TD><TD>0.819</TD></TR>
 +
<TR><TD>6</TD><TD>100</TD><TD>0.555</TD></TR>
 +
</TABLE>
 +
<p> To plot OD600 as a function of 2-nonenal concentration, I mixed 1mL of 2-nonenal with 5% of Tween80 (50uL). Then I prepared these solutions with a total volume of 1mL, and I measured their OD600 after 30min:
 +
<TABLE BORDER>
 +
<TR><TD><b>Tube</b></TD><TD><b>2-nonenal + Tween80 (uL)</b></TD><TD><b>Water (uL)</b></TD><TD><b>2-nonenal concentration (%tot. volume)</b></TD><TD><b>OD600(blank with Nuclease Free water)</b></TD></TR>
 +
<TR><TD>1</TD><TD>40</TD><TD>960</TD><TD>4%</TD><TD>1.957</TD></TR>
 +
<TR><TD>2</TD><TD>30</TD><TD>970</TD><TD>3%</TD><TD>1.318</TD></TR>
 +
<TR><TD>3</TD><TD>20</TD><TD>980</TD><TD>2%</TD><TD>1.265</TD></TR>
 +
<TR><TD>4</TD><TD>15</TD><TD>985</TD><TD>1.5%</TD><TD>0.667</TD></TR>
 +
<TR><TD>5</TD><TD>10</TD><TD>990</TD><TD>1%</TD><TD>0.504</TD></TR>
 +
<TR><TD>6</TD><TD>5</TD><TD>995</TD><TD>0.5%</TD><TD>0.344</TD></TR>
 +
</TABLE>
 +
</br>
 +
<p><u>3)Directed evolution</u></p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p> We started to have doubts about accuracy of the OD600 measurments because just by adding moe 2-nonenal to the media, we increase the density. However, with the measurments we did (2-nonenal + TWIN concentration vs. OD) we hope to estimate the cell content correctly. </p>
 +
<p> We modified the protocol. We measure OD600. We take 10um from of the overnight colony and we put it into the new medium with 5% more 2-nonenal and 5% less LB. This will allow us to see better growth of bacteria.</p>
 +
</br>
 +
                                        </br>
 +
                                        <h5 id="date">August 22th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 75%LB : 25% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<h5 id="date">August 23th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 70%LB : 30% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<h5 id="date">August 24th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.876</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 65%LB : 35% M9nonenal and put it into incubator 37C for overnight.</p>
 +
 +
                                        <h5 id="date">August 25th</h5>
 +
<p><u>1) Olfactive test experiment </u> </p>
 +
<p> This week we are leading our olfactive test experiment using sPB.050 and M9-2-nonenal medium. Each day of the week we start a liquid culture of sPB.050 in M9-2-nonenal: on Friday we will have 5 cultures, each one 0, 1, 2, 3 or 4 days old. We will plate each of this culture on LB to determine the CFU and have all the tubes smelled and rated by the team according to the intensity with a blind test.</p>
 +
</br>
 +
<p><u> 2)Plating the team's forehead samples</u> </p>
 +
<p> I plated on LB and on M9-2-nonenal Alexandre's, Sylvia's, Cristina's, Naresh's, Henry's and my forehead samples in order to find other bacteria developping on 2-nonenal.</p>
 +
<p><u>3) Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 60%LB : 40% M9nonenal and put it into incubator 37C for overnight.</p>
 +
 +
                                        <h5 id="date">August 26th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 55%LB : 45% M9nonenal and put it into incubator 37C for overnight.</p>
 +
                                        <h5 id="date">August 27th</h5>
 +
<p><u>1) Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:35% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 50%LB : 50% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<p>2) I got a suggestion from other member of the team that bacteria from the body samples growing on the LB plates in the experiment they  may differ depending on the way I put them on the plate. So far, we have been taking samples by scratching body surface with a loop. This time we took a cotton pad and inoculated it in LB in 37C overnight in order to plate them on 2-nonenal plates next day  </p>
 +
<p>3) Gram colouration. I performed the gram colouration in order identify the pSB.050. According to the observation the bacteria are gram negative (fuchsia colour).</p>
 +
 +
                                        <h5 id="date">August 28th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-0.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 45%LB : 55% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<p>The negative numbers may be surprising but taking into account that 2-nonenal with time get degraded (hopefully but bacteria in culture or by oxidation).</p>                                     
 +
 +
 +
<h5 id="date">August 29th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 40%LB : 60% M9nonenal and put it into incubator 37C for overnight.</p>
 +
 +
 +
<h5 id="date">August 30th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
<TR><TD>30.08 17h30</TD><TD>-1.551</TD><TD>-0.505</TD><TD>-1.495</TD><TD>0.299</TD><TD>40% LB:60% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 35%LB : 75% M9nonenal and put it into incubator 37C for overnight.</p>
 +
 +
 +
<div id="September">
<div id="September">
<h4>September</h4>
<h4>September</h4>
-
<h5 id="date">Date 1</h5>
+
<h5 id="date">September 1st</h5>
-
<p id="text">Text</p>
+
<p><u> Directed evolution</u></p>
-
<p id="text">Text</p>
+
<p>I measured OD600</p>
-
<h5 id="date">Date 2</h5>
+
                                      <TABLE BORDER>
-
<p id="text">Text</p>
+
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
-
<p id="text">Text</p>
+
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
<TR><TD>30.08 17h30</TD><TD>-1.551</TD><TD>-0.505</TD><TD>-1.495</TD><TD>0.299</TD><TD>40% LB:60% M9 2-nonenal</TD></TR>
 +
<TR><TD>01.09 12h00</TD><TD>-0.290</TD><TD>-0.056</TD><TD>-0.254</TD><TD>-0.055</TD><TD>35% LB:65% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 30%LB : 70% M9nonenal and put it into incubator 37C for overnight.</p>
 +
 
 +
<h5 id="date">September 2nd</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
<TR><TD>30.08 17h30</TD><TD>-1.551</TD><TD>-0.505</TD><TD>-1.495</TD><TD>0.299</TD><TD>40% LB:60% M9 2-nonenal</TD></TR>
 +
<TR><TD>01.09 12h00</TD><TD>-0.290</TD><TD>-0.056</TD><TD>-0.254</TD><TD>-0.055</TD><TD>35% LB:65% M9 2-nonenal</TD></TR>
 +
<TR><TD>02.09 12h10</TD><TD>-1.310</TD><TD>-0.728</TD><TD>-1.131</TD><TD>-1.180</TD><TD>30% LB:70% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 25%LB : 75% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<h5 id="date">September 3rd</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
<TR><TD>30.08 17h30</TD><TD>-1.551</TD><TD>-0.505</TD><TD>-1.495</TD><TD>0.299</TD><TD>40% LB:60% M9 2-nonenal</TD></TR>
 +
<TR><TD>01.09 12h00</TD><TD>-0.290</TD><TD>-0.056</TD><TD>-0.254</TD><TD>-0.055</TD><TD>35% LB:65% M9 2-nonenal</TD></TR>
 +
<TR><TD>02.09 12h10</TD><TD>-1.310</TD><TD>-0.728</TD><TD>-1.131</TD><TD>-1.180</TD><TD>30% LB:70% M9 2-nonenal</TD></TR>
 +
<TR><TD>03.09 12h15</TD><TD>-1.607</TD><TD>-0.768</TD><TD>-0.344</TD><TD>-1.964</TD><TD>25% LB:75% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 20%LB : 80% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<h5 id="date">September 4th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
<TR><TD>30.08 17h30</TD><TD>-1.551</TD><TD>-0.505</TD><TD>-1.495</TD><TD>0.299</TD><TD>40% LB:60% M9 2-nonenal</TD></TR>
 +
<TR><TD>01.09 12h00</TD><TD>-0.290</TD><TD>-0.056</TD><TD>-0.254</TD><TD>-0.055</TD><TD>35% LB:65% M9 2-nonenal</TD></TR>
 +
<TR><TD>02.09 12h10</TD><TD>-1.310</TD><TD>-0.728</TD><TD>-1.131</TD><TD>-1.180</TD><TD>30% LB:70% M9 2-nonenal</TD></TR>
 +
<TR><TD>03.09 12h15</TD><TD>-1.607</TD><TD>-0.768</TD><TD>-0.344</TD><TD>-1.964</TD><TD>25% LB:75% M9 2-nonenal</TD></TR>
 +
<TR><TD>04.09 11h50</TD><TD>-1.645</TD><TD>-0.898</TD><TD>-0.931</TD><TD>-1.603</TD><TD>20% LB:80% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 15%LB : 85% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<h5 id="date">September 4th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
<TR><TD>30.08 17h30</TD><TD>-1.551</TD><TD>-0.505</TD><TD>-1.495</TD><TD>0.299</TD><TD>40% LB:60% M9 2-nonenal</TD></TR>
 +
<TR><TD>01.09 12h00</TD><TD>-0.290</TD><TD>-0.056</TD><TD>-0.254</TD><TD>-0.055</TD><TD>35% LB:65% M9 2-nonenal</TD></TR>
 +
<TR><TD>02.09 12h10</TD><TD>-1.310</TD><TD>-0.728</TD><TD>-1.131</TD><TD>-1.180</TD><TD>30% LB:70% M9 2-nonenal</TD></TR>
 +
<TR><TD>03.09 12h15</TD><TD>-1.607</TD><TD>-0.768</TD><TD>-0.344</TD><TD>-1.964</TD><TD>25% LB:75% M9 2-nonenal</TD></TR>
 +
<TR><TD>04.09 11h50</TD><TD>-1.645</TD><TD>-0.898</TD><TD>-0.931</TD><TD>-1.603</TD><TD>20% LB:80% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>Then I took 10um from each sample and I put it into the medium 15%LB : 85% M9nonenal and put it into incubator 37C for overnight.</p>
 +
<h5 id="date">September 5th</h5>
 +
<p><u> Directed evolution</u></p>
 +
<p>I measured OD600</p>
 +
                                      <TABLE BORDER>
 +
<TR><TD></TD><TD colspan=4>Absorbance [OD600]</TD><TD></TD></TR>
 +
<TR><TD><b>date</b></TD><TD><b>Forehead 1</b></TD><TD><b>Forehead 2</b></TD><TD><b>Armpit</b></TD><TD><b>Nose</b></TD><TD><b>medium</b></TD></TR>
 +
<TR><TD>18.08 10h25</TD><TD>0</TD><TD>0</TD><TD>x</TD><TD>x</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
 +
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
 +
<TR><TD>22.08 11h30</TD><TD>2.23</TD><TD>2.43</TD><TD>2.099</TD><TD>1.907</TD><TD>80% LB:20% M9 2-nonenal</TD></TR>
 +
<TR><TD>23.08 14h10</TD><TD>1.492</TD><TD>1.035</TD><TD>1.107</TD><TD>1.735</TD><TD>75% LB:25% M9 2-nonenal</TD></TR>
 +
<TR><TD>24.08 16h10</TD><TD>1.53</TD><TD>1.867</TD><TD>1.437</TD><TD>2.385</TD><TD>70% LB:30% M9 2-nonenal</TD></TR>
 +
<TR><TD>25.08 11h30</TD><TD>1.604</TD><TD>1.873</TD><TD>1.329</TD><TD>2.143</TD><TD>65% LB:35% M9 2-nonenal</TD></TR>
 +
<TR><TD>26.08 14h00</TD><TD>0.609</TD><TD>1.283</TD><TD>0.501</TD><TD>0.487</TD><TD>60% LB:40% M9 2-nonenal</TD></TR>
 +
<TR><TD>27.08 15h50</TD><TD>0.898</TD><TD>0.847</TD><TD>0.942</TD><TD>0.874</TD><TD>55% LB:45% M9 2-nonenal</TD></TR>
 +
<TR><TD>28.08 11h40</TD><TD>-1.316</TD><TD>-0.038</TD><TD>-0.651</TD><TD>-0.011</TD><TD>50% LB:50% M9 2-nonenal</TD></TR>
 +
<TR><TD>29.08 16h30</TD><TD>-1.395</TD><TD>-0.465</TD><TD>-1.175</TD><TD>0.054</TD><TD>45% LB:55% M9 2-nonenal</TD></TR>
 +
<TR><TD>30.08 17h30</TD><TD>-1.551</TD><TD>-0.505</TD><TD>-1.495</TD><TD>0.299</TD><TD>40% LB:60% M9 2-nonenal</TD></TR>
 +
<TR><TD>01.09 12h00</TD><TD>-0.290</TD><TD>-0.056</TD><TD>-0.254</TD><TD>-0.055</TD><TD>35% LB:65% M9 2-nonenal</TD></TR>
 +
<TR><TD>02.09 12h10</TD><TD>-1.310</TD><TD>-0.728</TD><TD>-1.131</TD><TD>-1.180</TD><TD>30% LB:70% M9 2-nonenal</TD></TR>
 +
<TR><TD>03.09 12h15</TD><TD>-1.607</TD><TD>-0.768</TD><TD>-0.344</TD><TD>-1.964</TD><TD>25% LB:75% M9 2-nonenal</TD></TR>
 +
<TR><TD>04.09 11h50</TD><TD>-1.645</TD><TD>-0.898</TD><TD>-0.931</TD><TD>-1.603</TD><TD>20% LB:80% M9 2-nonenal</TD></TR>
 +
<TR><TD>05.09 11h50</TD><TD>-1.457</TD><TD>-0.955</TD><TD>-0.479</TD><TD>-1.279</TD><TD>15% LB:75% M9 2-nonenal</TD></TR>
 +
</TABLE>
 +
<p>As I had to leave the lab for one week I plated 100um from each sample on LB and 100% M9 nonenal plates to see if can get colonies</p>
 +
<h5 id="date">September 10th</h5>
 +
<p> I observed colonies on the plates of 100% nonenal and LB of forehed 1 sPB.58, forehead 2 sPB.60, nose sPB.059.
 +
<h5 id="date">September 11th</h5>
 +
<p>We striked for single colonies on LB plates of 3 new found colonies and sPB.050.</p>
 +
<h5 id="date">September 12th</h5>
 +
<p>We performed a PCR colony PCR with 16s specific primers following <a href="https://2014.igem.org/Team:Paris_Bettencourt/Protocols#prot7">a standart protocol for Colony PCR</a> and run 5ul of the sample on an electrophoresis gel. <p><img src='https://static.igem.org/mediawiki/2014/0/04/16Spcrpb.png' ><p>Strains:</br>
 +
1 sPB.050</br>
 +
2 sPB.058</br>
 +
3 sPB.059</br>
 +
4 sPB.060 </br>
 +
5 sPB.061</br>
 +
6 E. coli control</br></p>
 +
<h5 id="date">September 13th</h5>
 +
<p>We have sent the samples for sequencing</p>
 +
<h5 id="date">September 14th</h5>
 +
<p>I made agar plates LB + differents quantities of 2-nonenal ranging from 1% to 1/32% by 2 fold. To 50 ml of LB I added 0.59 ml for 1%, 0.29 ml for 1/2% etc. I had LB plates as a control. I made serial dilutions 10^2 10^4 and 10^6 of overnight culture of sPB.050 with PSB x10 and plated each ditlution on all different concentrations.</p>
 +
<h5 id="date">September 15th</h5>
 +
<p>CFU observed was as follows  <TABLE BORDER><TR><TD><b>2-nonenal conentration</b></TD><TD><b>10<sup>2</sup></b></TD><TD><b>10<sup>4</sup></b></TD><TD><b>10<sup>6</sup></b></TD></TR>
 +
<TR><TD>1%</TD><TD>0</TD><TD>0</TD><TD>0</TD></TR>
 +
<TR><TD>1/2%</TD><TD>0</TD><TD>0</TD><TD>0</TD></TR>
 +
<TR><TD>1/4%</TD><TD>0</TD><TD>0</TD><TD>0</TD></TR>
 +
<TR><TD>1/8%</TD><TD>0</TD><TD>0</TD><TD>0</TD></TR>
 +
<TR><TD>1/16%</TD><TD>+++</TD><TD>200</TD><TD>0</TD></TR>
 +
<TR><TD>1/32%</TD><TD>+++</TD><TD>240</TD><TD>1</TD></TR>
 +
<TR><TD>0%</TD><TD>+++</TD><TD>320</TD><TD>2</TD></TR>
 +
</TABLE>
 +
</p>
 +
<h5 id="date">September 25th</h5>
 +
<p>Smell test was prepared. 2-nonenal at 1% concentration (59uL per 5mL H<sub>2</sub>) was diltued 100 fold in each tube by putting 10ul on 1% into 990uL, then from 1/100% into 990uL etc etc. Thus we had dilutions 10<sup>2</sup>, 10<sup>4</sup>, 10<sup>6</sup>, 10<sup>8</sup>.
 +
<h5 id="date">September 30th</h5>
 +
<p>Isolated strains sPB.050, sPB.058, sPB.58 were innoculated in LB with 1/16% 2-nonenal and 0.05% tween to mix. I also prapared a control: LB with 1/16% 2-nonenal and 0.05% tween to mix and no bacteria.
 +
</p>
 +
 
</br>
</br>
</div>
</div>
<div id="October">
<div id="October">
<h4>October</h4>
<h4>October</h4>
-
<h5 id="date">Date 1</h5>
+
<h5 id="date">October 1st</h5>
-
<p id="text">Text</p>
+
<p id="text">I smelled the tubes and they didin't remarqued any smell difference </p>
-
<p id="text">Text</p>
+
 
-
<h5 id="date">Date 2</h5>
+
<h5 id="date">October 2nd</h5>
-
<p id="text">Text</p>
+
<p id="text">I smelled the tubes and remarqued a significant diffrence in the smell of sPB.050 il LB with noneal vs. control. There were no bacteria in the sample sPB.059. I preapred a smell test with the tubes sPB.050, sPB.058, sPB.059 </p>
-
<p id="text">Text</p>
+
</br>
</br>
</div>
</div>
</div>
</div>
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{{:Team:Paris_Bettencourt/Footer}}

Latest revision as of 03:12, 18 October 2014



Notebook


August


August 5th

The goal is to select a bacteria able to develop on 2-nonenal. We made 3 media:

1) M9 Minimal Media 100% Glucose

  • 200mL M9 Salts
  • 0.1mL CaCl2 1M
  • 2mL MgSO4 1M
  • 20mL Glucose 20%
  • Water: bring to 1L

2) M9 Minimal Media 50% Glucose - 50% 2-Nonenal (according to the number of carbon atoms)

  • 200mL M9 Salts
  • 0.1mL CaCl2 1M
  • 2mL MgSO4 1M
  • 10mL Glucose 20%
  • 10mL 2-nonenal 10,7%*
  • Water: bring to 1L

3) M9 Minimal Media 100% 2-nonenal

  • 200mL M9 Salts
  • 0.1mL CaCl2 1M
  • 2mL MgSO4 1M
  • 20mL 2-nonenal 10,7%*
  • Water: bring to 1L

We autoclaved 6 bottles (2 of 500mL for each)

Then, we made liquid culture of C.striatum in media 1) to try if the minima media works.


* Concentration of the 2-nonenal solution: In M9 Media, we use 20mL of Glucose 20% (200g/L). The concentration of glucose is C=200/M=1.09 mol/L. Glucose has 6 carbons while 2-nonenal has 9, so we need 2/3 of the glucose volume to have the same amount of carbon atoms. C=2/3*1.09=0.74 mol/L and then 0.74*M=103.7g/L. That's why the concentration of the 2-nonenal solution is 10.73%. We also added Tween80.



August 6th

We poured 20 plates of each of the 3 media with 350mL of an agar solution and 100mL of medium.

Then we used 3 plates of each type to culture skin bacteria from 3 spots: arm, armpit and forehead. Ursczula sampled bacteria from her skin with a loop and for each spot she diluted the bacteria in 300uL of NF Water. Then we plated 100uL of each solution in each of the 3 types of plates and incubated them at 37°C.

M9 100% glucoseM9 50%-50%M9 100% 2-nonenal
Armxxx
Armpitxxx
Foreheadxxx
NEB (control)xxx


August 8th

After 2 days, nothind has developped on the M9 plates. M9 media is actually really slow for the development of any strain. That is why we created a new media which would be like the real skin environment: synthetic sweat according to this recipe

According to the recipe, fatty acids seem to be the main source of carbon in synthetic sweat so we replaced them by 2-nonenal.
We made 2 media:
1) without fatty acids
2) without fatty acids and with 2-nonenal mixed with 10% of Tween 80.

August 11th

Results of M9 plates after 5 days

M9 100% glucoseM9 50%-50%M9 100% 2-nonenal
ArmMany very small colonies60
ArmpitMany very small colonies00
Forehead2750
NEB (control)Manymany0

Analysis:
The fitness of bacteria is better on 100% Glucose than on 50-50%. No colony develops on 2-nonenal, not even NEB. Then we can suggest that 2-nonenal is not used as a carbon source and that the difference of fitness between 100% glucose and 50-50% is only due to the lower amount of glucose in 50-50%. We should have made plates with only 50% of glucose to compare.


We also plated samples on synthetic sweat:

NEBForeheadNose
1) Without fatty acidsxxx
2) Without fatty acids and with 2-nonenalxxx

On August 10th, Jake tried a new medium with peptone added to M9. He made 2 media (20 plates of each):

  • iGEM medium 1 (100% glucose)
    • pancreatic digest of casein: 15 g
    • glucose: 5 g
    • NaCl: l5 g
    • Agar: 15 g
    • Water: 1 L
  • iGEM medium 2 (100% 2-nonenal):
    • pancreatic digest of casein: 15 g
    • 2-nonenal: 10 g
    • Tween 80: 250uL (premix with 2-nonenal before adding to the medium)
    • NaCl: l5 g
    • Agar: 15 g
    • Water: 1 L

I prepared 2 new media:

  • iGEM medium 3 (50% glucose)
    • pancreatic digest of casein: 7.5 g
    • glucose: 1.25 g
    • NaCl: 7.5 g
    • Agar: 7.5 g
    • Water: 0.5 L
  • iGEM medium 4 (50% glucose - 50% 2-nonenal):
    • pancreatic digest of casein: 7.5 g
    • glucose: 1.25 g
    • 2-nonenal: 2.5 g
    • Tween 80: 125uL (premix with 2-nonenal before adding to the autoclaved medium)
    • NaCl: 7.5 g
    • Agar: 7.5 g
    • Water: 0.5 L

I autoclaved them at 111°C - 30min (programm 5).

Then, I added the 2-nonenal to iGEM medium 4: density of nonenal is 0.846g/mL so I put 2.5/0.846 = 2.96 mL of 2-nonenal mixed with 125uL of Tween 80. I poured 20 plates of each medium and then plated before incubating at 37°C:

iGEM medium 1iGEM medium 2iGEM medium 3iGEM medium 4
Nosexxxx
Foreheadxxxx
NEB (control)xxxx


August 13th

Results of M9 plates after 7 days

M9 100% glucoseM9 50%-50%M9 100% 2-nonenal
ArmMany very small colonies17 including 6 big ones6 very small?
ArmpitMany very small coloniesAbout 20 very small1 or 2 really small?
Forehead3281
NEB (control)Manymany0

I plated on LBA the colony developped on 100% 2-nonenal from the Urszula's forehead to developp it.



August 14th

The colony developped on M9 100% 2-nonenal developped perfectly on LBA plates. It seems to be Staphylococcus aureus. I transfered colonies on 3 M9 100% 2-nonenal plates and I started 2 liquid cultures: one in LB and one in M9 100% 2-nonenal.



August 17th

We started new approach: Directed evolution of forehead, armpit and nose skin samples in the liquid medium
First two forehead samples in 37°C in medium 95% (4.75mL) LB and 5% M9 Minimal Media 100% 2-nonenal (0.25 mL)



August 18th

1) After four days of culture, I measured the absorbance of the liquid cultures of Staphylococcus aureus in LB and in M9 100% 2-nonenal medium.

A
LB1.647
M9 100% 2-nonenal medium0.109

S.aureus still develops on 2-nonenal but its fitness is much lower than on LB. I let it grow a few days more.


2) M9 plates from August 6th

It seems like we got small colonies on every 100% 2-nonenal plate (NEB, Forehead, Arm and Armpit). For each one, I prepared 2 liquid cultures: 1 LB and 1 M9 100% 2-nonenal.


3)Directed evolution

Absorbance has been measured for 2 tubes. A = 0 in both tubes. I will give it one more day


For an armpit and nose skin samples I proceeded similarly as started yesterday: incubated in 37°C in medium 95% (4.75mL) LB and 5% M9 Minimal Media 100% 2-nonenal (0.25 mL) with a sample from my armpit and another sample from my nose skin.



August 19th

1) Directed evolution - gradual approach

Samples 1 and 2 in 95%LB:5% M9 2-nonenal medium grew well and the new armpit and nose skin samples as well

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal

I centrifuged all 4 samples, discarded supernatant and changed the medium to 90% LB and 10% M9 2-nonenal


2) Staphylococcus aureus

I plated on LBA the forehead colonies from August 15th which had developped on 2-nonenal. Next week we will make a olfactive test to estimate the decrease of 2-nonenal using S.aureus. This strain will be called (N+)-S.aureus.

August 20th

1) Directed evolution - gradual approach

Corynebacterium striatum and nose and forehead samples density in 90%LB:10% M9 2-nonenal medium

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal

I centrifuged all 4 samples, discharged supernatant and changed the medium to 85% LB and 15% M9 2-nonenal


2) Plates (Samples 1 and 2)

The LB plate of (N+)-S.aureus has many colonies. I made a liquid culture to get a glycerol stock.

3) New M9

I prepared 2x50mL of each media:

  • M9 - 2-nonenal (120uL mixed for 3uL of Tween80 added after autoclave for 50mL of medium)
  • M9 - 2-nonenal (120uL mixed for 3uL of Tween80 added after autoclave for 50mL of medium) + 0.5g of peptone for 50mL of medium

I want to try to make M9 a faster medium. I started a liquid culture of (N+)-S.aureus and measured the initial density of each medium.

OD600
M9 - 2-nonenal0.885
M9 - 2-nonenal + peptone0.994


August 21th

1) Glycerol stock

I made a glycerol stock using (N+)-S.aureus liquid culture in LB according to this protocol. The strain is now called sPB.050

2) M9 overnight liquid cultures

After 24 hours, I measured the OD600 of the M9 liquid cultures with or without 2-nonenal.

OD600 before cultureOD600 after 24H culture
M9 - 2-nonenal0.8850.424
M9 - 2-nonenal + peptone0.9940.511

The result can be surprising because OD600 has decreased. But we figured out thant the main element which sets OD600 is the concentration of 2-nonenal because our medium is an emulsion. That is why OD600 is really variable according to the mixture. There is here a balance between the bacterian growth which increases OD600, correlated to a decrease of 2-nonenal concentration which lowers OD600 because it is consumed by the cells. It would be interesting to plot OD600 as a function of 2-nonenal concentration and CFU.

To plot OD600 as a function of CFU, I used the (N+)-S.aureus liquid culture in LB, putting different volumes in each tube. Then I centrifuged the tubes (1min-5000tr/min), discarded the supernatant, added 1mL of PBS and resuspended the cell by vortexing. I measured OD600 (with 500uL of solution) and then plated the solutions (100uL in each plate).

TubeVolume of LB culture (uL)OD600
110002.581
25001.758
32501.104
42000.927
51500.819
61000.555

To plot OD600 as a function of 2-nonenal concentration, I mixed 1mL of 2-nonenal with 5% of Tween80 (50uL). Then I prepared these solutions with a total volume of 1mL, and I measured their OD600 after 30min:

Tube2-nonenal + Tween80 (uL)Water (uL)2-nonenal concentration (%tot. volume)OD600(blank with Nuclease Free water)
1409604%1.957
2309703%1.318
3209802%1.265
4159851.5%0.667
5109901%0.504
659950.5%0.344

3)Directed evolution

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal

We started to have doubts about accuracy of the OD600 measurments because just by adding moe 2-nonenal to the media, we increase the density. However, with the measurments we did (2-nonenal + TWIN concentration vs. OD) we hope to estimate the cell content correctly.

We modified the protocol. We measure OD600. We take 10um from of the overnight colony and we put it into the new medium with 5% more 2-nonenal and 5% less LB. This will allow us to see better growth of bacteria.



August 22th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 75%LB : 25% M9nonenal and put it into incubator 37C for overnight.

August 23th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 70%LB : 30% M9nonenal and put it into incubator 37C for overnight.

August 24th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8761.4372.38570% LB:30% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 65%LB : 35% M9nonenal and put it into incubator 37C for overnight.

August 25th

1) Olfactive test experiment

This week we are leading our olfactive test experiment using sPB.050 and M9-2-nonenal medium. Each day of the week we start a liquid culture of sPB.050 in M9-2-nonenal: on Friday we will have 5 cultures, each one 0, 1, 2, 3 or 4 days old. We will plate each of this culture on LB to determine the CFU and have all the tubes smelled and rated by the team according to the intensity with a blind test.


2)Plating the team's forehead samples

I plated on LB and on M9-2-nonenal Alexandre's, Sylvia's, Cristina's, Naresh's, Henry's and my forehead samples in order to find other bacteria developping on 2-nonenal.

3) Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 60%LB : 40% M9nonenal and put it into incubator 37C for overnight.

August 26th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 55%LB : 45% M9nonenal and put it into incubator 37C for overnight.

August 27th

1) Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:35% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 50%LB : 50% M9nonenal and put it into incubator 37C for overnight.

2) I got a suggestion from other member of the team that bacteria from the body samples growing on the LB plates in the experiment they may differ depending on the way I put them on the plate. So far, we have been taking samples by scratching body surface with a loop. This time we took a cotton pad and inoculated it in LB in 37C overnight in order to plate them on 2-nonenal plates next day

3) Gram colouration. I performed the gram colouration in order identify the pSB.050. According to the observation the bacteria are gram negative (fuchsia colour).

August 28th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-0.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 45%LB : 55% M9nonenal and put it into incubator 37C for overnight.

The negative numbers may be surprising but taking into account that 2-nonenal with time get degraded (hopefully but bacteria in culture or by oxidation).

August 29th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 40%LB : 60% M9nonenal and put it into incubator 37C for overnight.

August 30th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal
30.08 17h30-1.551-0.505-1.4950.29940% LB:60% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 35%LB : 75% M9nonenal and put it into incubator 37C for overnight.

September

September 1st

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal
30.08 17h30-1.551-0.505-1.4950.29940% LB:60% M9 2-nonenal
01.09 12h00-0.290-0.056-0.254-0.05535% LB:65% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 30%LB : 70% M9nonenal and put it into incubator 37C for overnight.

September 2nd

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal
30.08 17h30-1.551-0.505-1.4950.29940% LB:60% M9 2-nonenal
01.09 12h00-0.290-0.056-0.254-0.05535% LB:65% M9 2-nonenal
02.09 12h10-1.310-0.728-1.131-1.18030% LB:70% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 25%LB : 75% M9nonenal and put it into incubator 37C for overnight.

September 3rd

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal
30.08 17h30-1.551-0.505-1.4950.29940% LB:60% M9 2-nonenal
01.09 12h00-0.290-0.056-0.254-0.05535% LB:65% M9 2-nonenal
02.09 12h10-1.310-0.728-1.131-1.18030% LB:70% M9 2-nonenal
03.09 12h15-1.607-0.768-0.344-1.96425% LB:75% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 20%LB : 80% M9nonenal and put it into incubator 37C for overnight.

September 4th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal
30.08 17h30-1.551-0.505-1.4950.29940% LB:60% M9 2-nonenal
01.09 12h00-0.290-0.056-0.254-0.05535% LB:65% M9 2-nonenal
02.09 12h10-1.310-0.728-1.131-1.18030% LB:70% M9 2-nonenal
03.09 12h15-1.607-0.768-0.344-1.96425% LB:75% M9 2-nonenal
04.09 11h50-1.645-0.898-0.931-1.60320% LB:80% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 15%LB : 85% M9nonenal and put it into incubator 37C for overnight.

September 4th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal
30.08 17h30-1.551-0.505-1.4950.29940% LB:60% M9 2-nonenal
01.09 12h00-0.290-0.056-0.254-0.05535% LB:65% M9 2-nonenal
02.09 12h10-1.310-0.728-1.131-1.18030% LB:70% M9 2-nonenal
03.09 12h15-1.607-0.768-0.344-1.96425% LB:75% M9 2-nonenal
04.09 11h50-1.645-0.898-0.931-1.60320% LB:80% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 15%LB : 85% M9nonenal and put it into incubator 37C for overnight.

September 5th

Directed evolution

I measured OD600

Absorbance [OD600]
dateForehead 1Forehead 2ArmpitNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal
22.08 11h302.232.432.0991.90780% LB:20% M9 2-nonenal
23.08 14h101.4921.0351.1071.73575% LB:25% M9 2-nonenal
24.08 16h101.531.8671.4372.38570% LB:30% M9 2-nonenal
25.08 11h301.6041.8731.3292.14365% LB:35% M9 2-nonenal
26.08 14h000.6091.2830.5010.48760% LB:40% M9 2-nonenal
27.08 15h500.8980.8470.9420.87455% LB:45% M9 2-nonenal
28.08 11h40-1.316-0.038-0.651-0.01150% LB:50% M9 2-nonenal
29.08 16h30-1.395-0.465-1.1750.05445% LB:55% M9 2-nonenal
30.08 17h30-1.551-0.505-1.4950.29940% LB:60% M9 2-nonenal
01.09 12h00-0.290-0.056-0.254-0.05535% LB:65% M9 2-nonenal
02.09 12h10-1.310-0.728-1.131-1.18030% LB:70% M9 2-nonenal
03.09 12h15-1.607-0.768-0.344-1.96425% LB:75% M9 2-nonenal
04.09 11h50-1.645-0.898-0.931-1.60320% LB:80% M9 2-nonenal
05.09 11h50-1.457-0.955-0.479-1.27915% LB:75% M9 2-nonenal

As I had to leave the lab for one week I plated 100um from each sample on LB and 100% M9 nonenal plates to see if can get colonies

September 10th

I observed colonies on the plates of 100% nonenal and LB of forehed 1 sPB.58, forehead 2 sPB.60, nose sPB.059.

September 11th

We striked for single colonies on LB plates of 3 new found colonies and sPB.050.

September 12th

We performed a PCR colony PCR with 16s specific primers following a standart protocol for Colony PCR and run 5ul of the sample on an electrophoresis gel.

Strains:
1 sPB.050
2 sPB.058
3 sPB.059
4 sPB.060
5 sPB.061
6 E. coli control

September 13th

We have sent the samples for sequencing

September 14th

I made agar plates LB + differents quantities of 2-nonenal ranging from 1% to 1/32% by 2 fold. To 50 ml of LB I added 0.59 ml for 1%, 0.29 ml for 1/2% etc. I had LB plates as a control. I made serial dilutions 10^2 10^4 and 10^6 of overnight culture of sPB.050 with PSB x10 and plated each ditlution on all different concentrations.

September 15th

CFU observed was as follows

2-nonenal conentration102104106
1%000
1/2%000
1/4%000
1/8%000
1/16%+++2000
1/32%+++2401
0%+++3202

September 25th

Smell test was prepared. 2-nonenal at 1% concentration (59uL per 5mL H2) was diltued 100 fold in each tube by putting 10ul on 1% into 990uL, then from 1/100% into 990uL etc etc. Thus we had dilutions 102, 104, 106, 108.

September 30th

Isolated strains sPB.050, sPB.058, sPB.58 were innoculated in LB with 1/16% 2-nonenal and 0.05% tween to mix. I also prapared a control: LB with 1/16% 2-nonenal and 0.05% tween to mix and no bacteria.


October

October 1st

I smelled the tubes and they didin't remarqued any smell difference

October 2nd

I smelled the tubes and remarqued a significant diffrence in the smell of sPB.050 il LB with noneal vs. control. There were no bacteria in the sample sPB.059. I preapred a smell test with the tubes sPB.050, sPB.058, sPB.059


Centre for Research and Interdisciplinarity (CRI)
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