Difference between revisions of "Team:Uppsala/Results"

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<hr>

−

<li><a class="tab" href="~~#enz_deg~~">Enzymatic degradation</a></li>

+

<li><a class="tab" href="https://2015.igem.org/Team:Uppsala/Results_enz">Enzymatic degradation</a></li>

−

<li><a class="tab" href="~~#naph~~">Naphthalene pathway</a></li>

+

<li><a class="tab" href="https://2015.igem.org/Team:Uppsala/Results_naph">Naphthalene pathway</a></li>

−

<li><a class="tab" href="~~#biosurf~~">Biosurfactants</a></li>

+

<li><a class="tab" href="https://2015.igem.org/Team:Uppsala/Results_bio">Biosurfactants</a></li>

</ul>

+

Please click on the links above to view the results for the different parts of our project.

−

<h2 id="enz_deg">Enzymatic degradation</h2>

+

<!-- <h2 id="enz_deg">Enzymatic degradation</h2>

<hr>

Restriction free cloning (RFC)

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<figcaption>9.3ABTS assay. The degradation of ABTS by the lysate of ModLac and the lysate of CueO over time.</figcaption></figcaption>

−

+

<tr>

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</table>

−

<figcaption>Figure 3: Gel electrophoresis. Well 1: cut <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> well 3: cut <a href="http://parts.igem.org/Part:BBa_K1688002">BBa_K1688002</a> and well 4: cut <a href="http://parts.igem.org/Part:BBa_K1688003">BBa_K1688003</a>. All biobricks cut with EcoRI and PstI. Well 2: DNA size marker commercial 1kb. 1% w/v agarose gel stained with SyberSafe.</figcaption>

+

<figcaption>Figure 1: Gel electrophoresis. Well 1: cut <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> well 3: cut <a href="http://parts.igem.org/Part:BBa_K1688002">BBa_K1688002</a> and well 4: cut <a href="http://parts.igem.org/Part:BBa_K1688003">BBa_K1688003</a>. All biobricks cut with EcoRI and PstI. Well 2: DNA size marker commercial 1kb. 1% w/v agarose gel stained with SyberSafe.</figcaption>

−

<figcaption>Figure 4: Gel electrophoresis. Well 11: cut <a href="http://parts.igem.org/Part:BBa_K1688001">BBa_K1688001</a> with XbaI and PstI. Well 8: DNA size marker 1kb. 1% w/v agarose gel stained with GelRed. </figcaption>

+

<figcaption>Figure 2: Gel electrophoresis. Well 11: cut <a href="http://parts.igem.org/Part:BBa_K1688001">BBa_K1688001</a> with XbaI and PstI. Well 8: DNA size marker 1kb. 1% w/v agarose gel stained with GelRed. </figcaption>

−

Figures 3 and 4 shows bands for each construct approximately as expected according to table 1. All biobrick constructs were verified by Sanger sequencing.

+

Figures 1 and 2 shows bands for each construct approximately as expected according to table 1. All biobrick constructs were verified by Sanger sequencing.

Verification of transcription of genes rhlA and rhlB with dTomato as reporter

−

<figcaption>Figure 5: E.coli DH5α transformed with assembled product <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> + <a href="http://parts.igem.org/Part:BBa_K1688004">BBa_K1688004</a>(dTomato construct) on agar plate.</figcaption>

+

<figcaption>Figure 3: E.coli DH5α transformed with assembled product <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> + <a href="http://parts.igem.org/Part:BBa_K1688004">BBa_K1688004</a>(dTomato construct) on agar plate.</figcaption>

−

Red fluorescent color expression of cells from figure 5 indicates that the mono-rhamnolipid gene construct is working, in effect the genes rhlA and rhlB are transcribed.

+

Red fluorescent color expression of cells from figure 3 indicates that the mono-rhamnolipid gene construct is working, in effect the genes rhlA and rhlB are transcribed.

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</table>

−

<figcaption>Figure 6: A bar graph displaying the expansion of drop in percentage of standard mono-rhamnolipids, 0, 0.2, 0.4, 0.6, 1, 1.6 mg/ml. Data from table 2</figcaption>

+

<figcaption>Figure 4: A bar graph displaying the expansion of drop in percentage of standard mono-rhamnolipids, 0, 0.2, 0.4, 0.6, 1, 1.6 mg/ml. Data from table 2</figcaption>

<figcaption>Table 3: Drop collapse test for different samples; negative controls LB medium, BL21DE3 and DH5α, <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3 and DH5α. Diameter of drop after 0,5,10,15 and 20 min, expansion of drop diameter in percentage and if the drop collapsed.</figcaption>

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−

<figcaption>Figure 7: A bar graph displaying the expansion of drop of different samples. Data from table 3</figcaption>

+

<figcaption>Figure 5: A bar graph displaying the expansion of drop of different samples. Data from table 3</figcaption>

−

Table 2 and figure 6 displays data of drop expansion test with standard mono-rhamnolipids (0, 0.2, 0.4, 0.6, 1 and 1.6 mg/ml). Table 3 and figure 7 displays the data of drop expansion test of LB medium, supernatant extracted from E.coli BL21DE3 with <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> respectively untransformed and supernatant extracted from E.coli DH5α with <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> respectively untransformed.

+

Table 2 and figure 4 displays data of drop expansion test with standard mono-rhamnolipids (0, 0.2, 0.4, 0.6, 1 and 1.6 mg/ml). Table 3 and figure 5 displays the data of drop expansion test of LB medium, supernatant extracted from E.coli BL21DE3 with <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> respectively untransformed and supernatant extracted from E.coli DH5α with <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> respectively untransformed.

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CTAB

−

<figcaption>Figure 8: in E.coli BL21DE3 cells with <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> on CTAB plate.</figcaption>

+

<figcaption>Figure 6: in E.coli BL21DE3 cells with <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> on CTAB plate.</figcaption>

−

The appearance of halos around the colonies on CTAB plates, figure 8 indicates the expression of rhamnolipids.

+

The appearance of halos around the colonies on CTAB plates, figure 6 indicates the expression of rhamnolipids.

TLC

−

<figcaption>Figure 9: TLC silica plates stained with a orcinol-sulphuric acid solution. From lane 1 to 6: BL21DE3 untransformed, <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3, P.Putida and standard mono-rhamnolipids 10, 30 and 50 μg.</figcaption>

+

<figcaption>Figure 7: TLC silica plates stained with a orcinol-sulphuric acid solution. From lane 1 to 6: BL21DE3 untransformed, <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3, P.Putida and standard mono-rhamnolipids 10, 30 and 50 μg.</figcaption>

<figcaption>Table 4: Retention factor (Rf) of different samples run on TLC silica plate.</figcaption>

<table>

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</table>

−

Clear spots were detected in lane 2, 4, 5 and 6 in figure 9 corresponding to the sample extracted from BL21DE3 cells with biobrick <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> and standard mono-rhamnolipid 10, 30 respectively 50 μg. The detection spot of <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> had a retention factor 0,82, the same or similar retention factor as the detection spots for standard mono-rhamnolipids (table 4), which confirms mono-rhamnolipid synthesis by <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3 cells.

+

Clear spots were detected in lane 2, 4, 5 and 6 in figure 7 corresponding to the sample extracted from BL21DE3 cells with biobrick <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> and standard mono-rhamnolipid 10, 30 respectively 50 μg. The detection spot of <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> had a retention factor 0,82, the same or similar retention factor as the detection spots for standard mono-rhamnolipids (table 4), which confirms mono-rhamnolipid synthesis by <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3 cells.

−

Negative control; BL21DE3 untransformed in lane 1 (figure 9) showed no spot which is expected as BL21DE3 do not produce biosurfactants naturally. P. putida as a positive control showed no spot. This might be because of too low concentration of rhamnolipids in sample, problems with extraction of rhamnolipids or sample contamination. Low concentration of rhamnolipids in supernatant might be because of used medium and growth conditions.

+

Negative control; BL21DE3 untransformed in lane 1 (figure 7) showed no spot which is expected as BL21DE3 do not produce biosurfactants naturally. P. putida as a positive control showed no spot. This might be because of too low concentration of rhamnolipids in sample, problems with extraction of rhamnolipids or sample contamination. Low concentration of rhamnolipids in supernatant might be because of used medium and growth conditions.

Mass spectrometry

−

<figcaption>Figure 10: The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with RhlA and RhlB gene (<a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>). The m/z 447 ion chromatogram corresponding to (M-H)- of Rha-C8-C8 (mono-rhamnolipid) with retention time at 2.82. </figcaption>

+

<figcaption>Figure 8: The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with RhlA and RhlB gene (<a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>). The m/z 447 ion chromatogram corresponding to (M-H)- of Rha-C8-C8 (mono-rhamnolipid) with retention time at 2.82. </figcaption>

−

<figcaption>Figure 11: The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with RhlA and RhlB gene (<a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>). The m/z 503 ion chromatogram corresponding to (M-H)- of Rha-C10-C10 (mono-rhamnolipid) with retention time at 4.08.</figcaption>

+

<figcaption>Figure 9: The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with RhlA and RhlB gene (<a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>). The m/z 503 ion chromatogram corresponding to (M-H)- of Rha-C10-C10 (mono-rhamnolipid) with retention time at 4.08.</figcaption>

−

<figcaption>Figure 12: The mass spectrum from total ion chromatogram (TIC) of lipid extraction of E.coli BL21DE3 with RhlA and RhlB gene (<a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>).</figcaption>

+

<figcaption>Figure 10: The mass spectrum from total ion chromatogram (TIC) of lipid extraction of E.coli BL21DE3 with RhlA and RhlB gene (<a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>).</figcaption>

−

Figure 10-12 shows result for mass spectrometry of lipid extraction of E.coli BL21DE3 expressing biobrick <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>. Figure 10 indicates presence of mono-rhamnolipid type Rha-C8-C8 in the sample. Figure 11 and 12 indicates presence of mono-rhamnolipid type Rha-C10-C10 in the sample.

+

Figure 8-10 shows result for mass spectrometry of lipid extraction of E.coli BL21DE3 expressing biobrick <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>. Figure 8 indicates presence of mono-rhamnolipid type Rha-C8-C8 in the sample. Figure 9 and 10 indicates presence of mono-rhamnolipid type Rha-C10-C10 in the sample.

Conclusion

The drop collapse test, CTAB test, TLC and mass spectrometry showed positive result and we could confirm that mono-rhamnolipids are expressed by our construct (<a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a>) with E.coli BL21DE3. However, we still need to study their expression in the presence of PAH degrading enzymes (dioxygenase and laccase) and PAHs, to know whether these may influence the mono-rhamnolipid synthesis. Our future plan is that biosurfactant strains will be used together with the strains that expresses the PAH degrading enzymes. The biosurfactants will break down the clustered PAHs and make them available to degrading enzymes for an efficient degradation.

−

+

-->

</div>

@@ Line 87: / Line 87: @@
    <hr>
    <ul id="tab_list">
-       <li><a class="tab" href="#enz_deg"><b>Enzymatic degradation</b></a></li>
+       <li><a class="tab" href="https://2015.igem.org/Team:Uppsala/Results_enz"><b>Enzymatic degradation</b></a></li>
-       <li><a class="tab" href="#naph"><b>Naphthalene pathway</b></a></li>
+       <li><a class="tab" href="https://2015.igem.org/Team:Uppsala/Results_naph"><b>Naphthalene pathway</b></a></li>
-       <li><a class="tab" href="#biosurf"><b>Biosurfactants</b></a></li>
+       <li><a class="tab" href="https://2015.igem.org/Team:Uppsala/Results_bio"><b>Biosurfactants</b></a></li>
     </ul>
+  <p>Please click on the links above to view the results for the different parts of our project.</p>
-  <h2 id="enz_deg">Enzymatic degradation</h2>
+ <!-- <h2 id="enz_deg">Enzymatic degradation</h2>
    <hr>
    <u><b><p>Restriction free cloning (RFC)</p></b></u>
@@ Line 185: / Line 186: @@
    <img src="https://static.igem.org/mediawiki/2015/8/82/Uppsala_figur9resultatgrp1.png">
    <figcaption><b>9.3ABTS assay. The degradation of ABTS by the lysate of ModLac and the lysate of CueO over time.</figcaption></b></figcaption>
-   <table style="border:none;">
+   <table style="border:none;width:700px;">
     <tr>
      <td style="border:none;"><img src="https://static.igem.org/mediawiki/2015/5/50/Uppsala_sds_1.png"></td>
@@ Line 361: / Line 362: @@
 </table>
    <img src="https://static.igem.org/mediawiki/2015/3/3a/Uppsala_fig3_bio.png">
-   <figcaption><b>Figure 3</b>: Gel electrophoresis. Well 1: cut <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> well 3: cut <a href="http://parts.igem.org/Part:BBa_K1688002"><span class="res_link">BBa_K1688002</span></a> and well 4: cut <a href="http://parts.igem.org/Part:BBa_K1688003"><span class="res_link">BBa_K1688003</span></a>. All biobricks cut with EcoRI and PstI. Well 2: DNA size marker commercial 1kb. 1% w/v agarose gel stained with SyberSafe.</figcaption>
+   <figcaption><b>Figure 1</b>: Gel electrophoresis. Well 1: cut <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> well 3: cut <a href="http://parts.igem.org/Part:BBa_K1688002"><span class="res_link">BBa_K1688002</span></a> and well 4: cut <a href="http://parts.igem.org/Part:BBa_K1688003"><span class="res_link">BBa_K1688003</span></a>. All biobricks cut with EcoRI and PstI. Well 2: DNA size marker commercial 1kb. 1% w/v agarose gel stained with SyberSafe.</figcaption>
    <img src="https://static.igem.org/mediawiki/2015/8/8d/Uppsala_fig4_bio.png">
-   <figcaption><b>Figure 4</b>: Gel electrophoresis. Well 11: cut <a href="http://parts.igem.org/Part:BBa_K1688001"><span class="res_link">BBa_K1688001</span></a> with XbaI and PstI. Well 8: DNA size marker 1kb. 1% w/v agarose gel stained with GelRed.  </figcaption>
+   <figcaption><b>Figure 2</b>: Gel electrophoresis. Well 11: cut <a href="http://parts.igem.org/Part:BBa_K1688001"><span class="res_link">BBa_K1688001</span></a> with XbaI and PstI. Well 8: DNA size marker 1kb. 1% w/v agarose gel stained with GelRed.  </figcaption>
    <p>
-   Figures 3 and 4 shows bands for each construct approximately as expected according to table 1. All biobrick constructs were verified by Sanger sequencing.
+   Figures 1 and 2 shows bands for each construct approximately as expected according to table 1. All biobrick constructs were verified by Sanger sequencing.
    </p>
    <u><b><p>Verification of transcription of genes <i>rhlA</i> and <i>rhlB</i> with dTomato as reporter</p></b></u>
    <img src="https://static.igem.org/mediawiki/2015/9/95/Uppsala_fig5_bio.png">
-   <figcaption><b>Figure 5</b>: <i>E.coli</i> DH5α transformed with assembled product <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> + <a href="http://parts.igem.org/Part:BBa_K1688004"><span class="res_link">BBa_K1688004</span></a>(dTomato construct) on agar plate.</figcaption>
+   <figcaption><b>Figure 3</b>: <i>E.coli</i> DH5α transformed with assembled product <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> + <a href="http://parts.igem.org/Part:BBa_K1688004"><span class="res_link">BBa_K1688004</span></a>(dTomato construct) on agar plate.</figcaption>
    <p>
-   Red fluorescent color expression of cells from figure 5 indicates that the mono-rhamnolipid gene construct is working, in effect the genes <i>rhlA</i> and <i>rhlB</i> are transcribed.
+   Red fluorescent color expression of cells from figure 3 indicates that the mono-rhamnolipid gene construct is working, in effect the genes <i>rhlA</i> and <i>rhlB</i> are transcribed.
    </p>
@@ Line 452: / Line 453: @@
    </table>
    <img src="https://static.igem.org/mediawiki/2015/9/9b/Uppsala_fig6_bio.png">
-   <figcaption><b>Figure 6</b>: A bar graph displaying the expansion of drop in percentage of standard mono-rhamnolipids, 0, 0.2, 0.4, 0.6, 1, 1.6 mg/ml. Data from table 2</figcaption>
+   <figcaption><b>Figure 4</b>: A bar graph displaying the expansion of drop in percentage of standard mono-rhamnolipids, 0, 0.2, 0.4, 0.6, 1, 1.6 mg/ml. Data from table 2</figcaption>
    <figcaption><b>Table 3</b>: Drop collapse test for different samples; negative controls LB medium, BL21DE3 and DH5α, <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> in BL21DE3 and DH5α.  Diameter of drop after 0,5,10,15 and 20 min, expansion of drop diameter in percentage and if the drop collapsed.</figcaption>
@@ Line 523: / Line 524: @@
    <img src="https://static.igem.org/mediawiki/2015/5/53/Uppsala_fig7_bio.png">
-   <figcaption><b>Figure 7</b>: A bar graph displaying the expansion of drop of different samples. Data from table 3</figcaption>
+   <figcaption><b>Figure 5</b>: A bar graph displaying the expansion of drop of different samples. Data from table 3</figcaption>
    <p>
-   Table 2 and figure 6 displays data of drop expansion test with standard mono-rhamnolipids (0, 0.2, 0.4, 0.6, 1 and 1.6 mg/ml). Table 3 and figure 7 displays the data of drop expansion test of LB medium, supernatant extracted from <i>E.coli</i> BL21DE3 with <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> respectively untransformed and supernatant extracted from <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> respectively untransformed.
+   Table 2 and figure 4 displays data of drop expansion test with standard mono-rhamnolipids (0, 0.2, 0.4, 0.6, 1 and 1.6 mg/ml). Table 3 and figure 5 displays the data of drop expansion test of LB medium, supernatant extracted from <i>E.coli</i> BL21DE3 with <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> respectively untransformed and supernatant extracted from <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> respectively untransformed.
    </p>
    <p>
@@ Line 533: / Line 534: @@
    <u><b><p>CTAB</p></b></u>
    <img src="https://static.igem.org/mediawiki/2015/e/e2/Uppsala_fig8_bio.png">
-   <figcaption><b>Figure 8</b>: in <i>E.coli</i> BL21DE3 cells with  <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> on CTAB plate.</figcaption>
+   <figcaption><b>Figure 6</b>: in <i>E.coli</i> BL21DE3 cells with  <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> on CTAB plate.</figcaption>
    <p>
-   The appearance of halos around the colonies on CTAB plates, figure 8 indicates the expression of rhamnolipids.
+   The appearance of halos around the colonies on CTAB plates, figure 6 indicates the expression of rhamnolipids.
    </p>
    <u><b><p>TLC</p></b></u>
    <img src="https://static.igem.org/mediawiki/2015/0/0b/Uppsala_fig8_bios.png">
-   <figcaption><b>Figure 9</b>: TLC silica plates stained with a orcinol-sulphuric acid solution. From lane 1 to 6: BL21DE3 untransformed, <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> in BL21DE3, <i>P.Putida</i> and standard mono-rhamnolipids 10, 30 and 50 μg.</figcaption>
+   <figcaption><b>Figure 7</b>: TLC silica plates stained with a orcinol-sulphuric acid solution. From lane 1 to 6: BL21DE3 untransformed, <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> in BL21DE3, <i>P.Putida</i> and standard mono-rhamnolipids 10, 30 and 50 μg.</figcaption>
    <figcaption><b>Table 4</b>: Retention factor (Rf) of different samples run on TLC silica plate.</figcaption>
    <table>
@@ Line 594: / Line 595: @@
    </table>
    <p>
-   Clear spots were detected in lane 2, 4, 5 and 6 in figure 9 corresponding to the sample extracted from BL21DE3 cells with biobrick <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> and standard mono-rhamnolipid 10, 30 respectively 50 μg. The detection spot of <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> had a retention factor 0,82, the same or similar retention factor as the detection spots for standard mono-rhamnolipids (table 4), which confirms mono-rhamnolipid synthesis by <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> in BL21DE3 cells.
+   Clear spots were detected in lane 2, 4, 5 and 6 in figure 7 corresponding to the sample extracted from BL21DE3 cells with biobrick <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> and standard mono-rhamnolipid 10, 30 respectively 50 μg. The detection spot of <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> had a retention factor 0,82, the same or similar retention factor as the detection spots for standard mono-rhamnolipids (table 4), which confirms mono-rhamnolipid synthesis by <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> in BL21DE3 cells.
    </p>
    <p>
-   Negative control; BL21DE3 untransformed in lane 1 (figure 9) showed no spot which is expected as BL21DE3 do not produce biosurfactants naturally. <i>P. putida</i> as a positive control showed no spot. This might be because of too low concentration of rhamnolipids in sample, problems with extraction of rhamnolipids or sample contamination. Low concentration of rhamnolipids in supernatant might be because of used medium and growth conditions.
+   Negative control; BL21DE3 untransformed in lane 1 (figure 7) showed no spot which is expected as BL21DE3 do not produce biosurfactants naturally. <i>P. putida</i> as a positive control showed no spot. This might be because of too low concentration of rhamnolipids in sample, problems with extraction of rhamnolipids or sample contamination. Low concentration of rhamnolipids in supernatant might be because of used medium and growth conditions.
    </p>
    <u><b><p>Mass spectrometry</p></b></u>
    <img src="https://static.igem.org/mediawiki/2015/9/92/Uppsala_fig10_bio.png">
-   <figcaption><b>Figure 10:</b> The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with <i>Rhl</i>A and <i>Rhl</i>B gene (<a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>). The m/z 447 ion chromatogram corresponding to (M-H)- of Rha-C8-C8 (mono-rhamnolipid) with retention time at 2.82. </figcaption>
+   <figcaption><b>Figure 8:</b> The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with <i>Rhl</i>A and <i>Rhl</i>B gene (<a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>). The m/z 447 ion chromatogram corresponding to (M-H)- of Rha-C8-C8 (mono-rhamnolipid) with retention time at 2.82. </figcaption>
    <img src="https://static.igem.org/mediawiki/2015/1/12/Uppsala_fig11_bio.png">
-   <figcaption><b>Figure 11:</b> The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with <i>Rhl</i>A and <i>Rhl</i>B gene (<a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>). The m/z 503 ion chromatogram corresponding to (M-H)- of Rha-C10-C10 (mono-rhamnolipid) with retention time at 4.08.</figcaption>
+   <figcaption><b>Figure 9:</b> The MRM chromatogram of the lipid extraction of E.coli BL21DE3 with <i>Rhl</i>A and <i>Rhl</i>B gene (<a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>). The m/z 503 ion chromatogram corresponding to (M-H)- of Rha-C10-C10 (mono-rhamnolipid) with retention time at 4.08.</figcaption>
    <img src="https://static.igem.org/mediawiki/2015/0/04/Uppsala_fig12_bio.png">
-   <figcaption><b>Figure 12:</b> The mass spectrum from total ion chromatogram (TIC) of lipid extraction of E.coli BL21DE3 with <i>Rhl</i>A and <i>Rhl</i>B gene (<a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>).</figcaption>
+   <figcaption><b>Figure 10:</b> The mass spectrum from total ion chromatogram (TIC) of lipid extraction of E.coli BL21DE3 with <i>Rhl</i>A and <i>Rhl</i>B gene (<a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>).</figcaption>
    <p>
-   Figure 10-12 shows result for mass spectrometry of lipid extraction of  E.coli BL21DE3 expressing biobrick <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>. Figure 10 indicates presence of mono-rhamnolipid type Rha-C8-C8 in the sample. Figure 11 and 12 indicates presence of mono-rhamnolipid type Rha-C10-C10 in the sample.
+   Figure 8-10 shows result for mass spectrometry of lipid extraction of  E.coli BL21DE3 expressing biobrick <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>. Figure 8 indicates presence of mono-rhamnolipid type Rha-C8-C8 in the sample. Figure 9 and 10 indicates presence of mono-rhamnolipid type Rha-C10-C10 in the sample.
    </p>
    <u><b><p>Conclusion</p></b></u>
    <p>
    The drop collapse test, CTAB test, TLC and mass spectrometry showed positive result and we could confirm that mono-rhamnolipids are expressed by our construct (<a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a>) with <i>E.coli</i> BL21DE3. However, we still need to study their expression in the presence of PAH degrading enzymes (dioxygenase and laccase) and PAHs, to know whether these may influence the mono-rhamnolipid synthesis. Our future plan is that biosurfactant strains will be used together with the strains that expresses the PAH degrading enzymes. The biosurfactants will break down the clustered PAHs and make them available to degrading enzymes for an efficient degradation.
-   </p>
+   </p>-->
   </div>