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<hr>

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<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>Figure 3: Agarose gel with PCR product from colony PCR of RFC product from the second PCR. Well 5 contains dioxygenase without the HlyA-tag attached and well 4 contains the RFC product which should have the HlyA-tag attached. The band in well 5 has moved further than the band in well 4 which shows that the PCR product in well 4 is longer than the product in well 5. Well 2 and 3 contains CueO with and without the HlyA-tag respectively. There is a difference in length between these two bands as well and this shows that the RFC was successful also for CueO.</figcaption>

−

Samples were sent for Sanger sequencing to SciLifeLab in Uppsala to confirm correct assembly of the HlyA-tag. Results ~~confirmed~~ that a 2 base frameshift in the coding sequence was introduced due to faulty primer design. This resulted in incorrect expression of the export tag, which made the tag useless.

+

Samples were sent for Sanger sequencing to SciLifeLab in Uppsala to confirm correct assembly of the HlyA-tag. Results showed that a 2 base frameshift in the coding sequence was introduced due to faulty primer design. This resulted in incorrect expression of the export tag, which made the tag useless.

+

Purification and characterization of catechol-1,2-dioxygenase

Conditions of cell lysis proved effective, with 100% of cells lysed as estimated by visual inspection. After centrifugation and filtration, the crude cell extract showed 12.47±2.31 mg/ml, as measured by NanoDrop at 280 nm. The total volume of the cell extract was around 60 ml.

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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>

+

+

+

</tr>

+

</table>

+

<figcaption>Figure 9.4: SDS-PAGE results of the IMAC fractions of the modified laccase (left) and CueO (right)</figcaption>

+

−

<figcaption>Figure 9.4: shows 9 fractions from Ion exchange column; one band of 20-30kDa.~~~~ </figcaption>

+

<figcaption>Figure 9.5: shows 9 fractions from Ion exchange column; one band of 20-30kDa. </figcaption>

NahR characterization

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IMAC

−

The chromatograms of ModLac and CueO (Ecol) showed very low protein concentrations (see Figures 9.1 and 9.2). The eluate fractions from the IMAC did not show any enzyme activity. Examination of these fractions with SDS-PAGE (see Figure Q) also showed that there were no, or very low concentrations of protein in them. This raises the question as to what went wrong in the purification method. The theory of IMAC rests upon the fact that his-tagged proteins will bind to the column and not elute until a high concentration of imidazole is pumped through the column. Enzyme activity was found in the lysate of CueO and its mutant, which means that there were functioning proteins prior to the IMAC. Possible explanations as to why no activity was measured in the eluate could be that the protein concentration was too low in the elution fraction (due to dilution) or that no protein bound to the column. This was confirmed when enzyme activity was found in the initial wash of the column. These results point toward the fact that something went wrong with the interaction between the protein and the column. It is unlikely that the nickel ions had not bound to the gel, since the columns had a clear light turquoise colour after being loaded. Also, the colour was not washed away during the IMAC procedure. There is a risk that the polyhistidine-tag, that was fused to the proteins, had folded into the protein and was not exposed on the protein surface. The tag needs to be on the surface of the protein to enable interaction with the nickel-enriched gel. This could have been checked by denaturing the protein samples prior to the IMAC, which would ensure that the his-tag was fully unfolded. This would require renaturation of the protein to be able to measure enzyme activity however.

+

The chromatograms of ModLac and CueO (Ecol) showed very low protein concentrations (see Figures 9.1 and 9.2). The eluate fractions from the IMAC did not show any enzyme activity. Examination of these fractions with SDS-PAGE (see Figure 9.4) also showed that there were no, or very low concentrations of protein in them. This raises the question as to what went wrong in the purification method. The theory of IMAC rests upon the fact that his-tagged proteins will bind to the column and not elute until a high concentration of imidazole is pumped through the column. Enzyme activity was found in the lysate of CueO and its mutant, which means that there were functioning proteins prior to the IMAC. Possible explanations as to why no activity was measured in the eluate could be that the protein concentration was too low in the elution fraction (due to dilution) or that no protein bound to the column. This was confirmed when enzyme activity was found in the initial wash of the column. These results point toward the fact that something went wrong with the interaction between the protein and the column. It is unlikely that the nickel ions had not bound to the gel, since the columns had a clear light turquoise colour after being loaded. Also, the colour was not washed away during the IMAC procedure. There is a risk that the polyhistidine-tag, that was fused to the proteins, had folded into the protein and was not exposed on the protein surface. The tag needs to be on the surface of the protein to enable interaction with the nickel-enriched gel. This could have been checked by denaturing the protein samples prior to the IMAC, which would ensure that the his-tag was fully unfolded. This would require renaturation of the protein to be able to measure enzyme activity however.

ABTS assay on ModLac and CueO

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−

The SDS-PAGE (Figure 9.4) showed that we did not manage to purify the CueO D439A/M510L mutant, though the NanoDrop results shows that there should be 11 g/ml of the protein. The SDS-PAGE and Nanodrop relative to each other we can establish that the NanoDrop results are unreliable.

+

The SDS-PAGE (Figure 9.5) showed that we did not manage to purify the CueO D439A/M510L mutant, though the NanoDrop results shows that there should be 11 g/ml of the protein. The SDS-PAGE and Nanodrop relative to each other we can establish that the NanoDrop results are unreliable.

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−

The probable source of error is the buffer. The SDS-PAGE (Figure ~~LOL IEC~~)results showed that it can be carbonic anhydrase (Can), that is produced during stress and starvation in the BL21DE3 strain and has a pI of 6.4. The overnight had been in the cold room for some time and had started to show floating webs of colonies.

+

The probable source of error is the buffer. The SDS-PAGE (Figure 9.5)results showed that it can be carbonic anhydrase (Can), that is produced during stress and starvation in the BL21DE3 strain and has a pI of 6.4. The overnight had been in the cold room for some time and had started to show floating webs of colonies.

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</tr>

<tr>

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+

<td>EcoRI, PstI</td>

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</table>

−

<figcaption>Figure 3: Gel electrophoresis. Well 1: cut BBa_K1688000, well 3: cut BBa_K1688002 and well 4: cut BBa_K1688003. 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 BBa_K1688001 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 BBa_K1688000 + ~~BBa_1688004~~ (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, BBa_K1688000 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>

+

<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>

<table>

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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 BBa_K1688000 respectively untransformed and supernatant extracted from E.coli DH5α with BBa_K1688000 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.

−

Table 2 shows that a higher concentration of mono-rhamnolipids causes the drop to expand more and collapse faster. This verifies that presence of rhamnolipids can be indicated from drop collapse tests. The drop from sample BBa_K1688000 in BL21DE3 from table 3 collapsed after 30 seconds and expansion of drop diameter was 120% within 5 minutes from 1 cm to 2.2 cm which indicate presence of biosurfactant. The drop from sample BBa_K1688000 in DH5α collapsed and diameter expansion of drop was 90% after 20 minutes. This indicates some presence of biosurfactants. As expected the test indicate that BBa_K1688000 has higher expression rates and rhamnolipid production was higher in BL21DE3 than in DH5α as BL21DE3 is good for protein expression. The negative controls, LB medium and untransformed BL21DE3 and DH5α showed very little expansion or no expansion, which is expected as they do not produce biosurfactants.

+

Table 2 shows that a higher concentration of mono-rhamnolipids causes the drop to expand more and collapse faster. This verifies that presence of rhamnolipids can be indicated from drop collapse tests. The drop from sample <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3 from table 3 collapsed after 30 seconds and expansion of drop diameter was 120% within 5 minutes from 1 cm to 2.2 cm which indicate presence of biosurfactant. The drop from sample <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in DH5α collapsed and diameter expansion of drop was 90% after 20 minutes. This indicates some presence of biosurfactants. As expected the test indicate that <a href="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> has higher expression rates and rhamnolipid production was higher in BL21DE3 than in DH5α as BL21DE3 is good for protein expression. The negative controls, LB medium and untransformed BL21DE3 and DH5α showed very little expansion or no expansion, which is expected as they do not produce biosurfactants.

CTAB

−

<figcaption>Figure 8: in E.coli BL21DE3 cells with BBa_K1688000 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, BBa_K1688000 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 BBa_K1688000 and standard mono-rhamnolipid 10, 30 respectively 50 μg. The detection spot of BBa_K1688000 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 BBa_K1688000 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 (BBa_K1688000). 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 (BBa_K1688000). 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 (BBa_K1688000).</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 BBa_K1688000. 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 (BBa_K1688000) 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.

+

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 112: / Line 113: @@
    <figcaption><b>Figure 3</b>: Agarose gel with PCR product from colony PCR of RFC product from the second PCR. Well 5 contains dioxygenase without the HlyA-tag attached and well 4 contains the RFC product which should have the HlyA-tag attached. The band in well 5 has moved further than the band in well 4 which shows that the PCR product in well 4 is longer than the product in well 5. Well 2 and 3 contains CueO with and without the HlyA-tag respectively. There is a difference in length between these two bands as well and this shows that the RFC was successful also for CueO.</figcaption>
    <p>
-   Samples were sent for Sanger sequencing to SciLifeLab in Uppsala to confirm correct assembly of the HlyA-tag. Results confirmed that a 2 base frameshift in the coding sequence was introduced due to faulty primer design. This resulted in incorrect expression of the export tag, which made the tag useless.
+   Samples were sent for Sanger sequencing to SciLifeLab in Uppsala to confirm correct assembly of the HlyA-tag. Results showed that a 2 base frameshift in the coding sequence was introduced due to faulty primer design. This resulted in incorrect expression of the export tag, which made the tag useless.
    </p>
+<p><b><u>Purification and characterization of catechol-1,2-dioxygenase</p></b></u>
    <p>
    Conditions of cell lysis proved effective, with 100% of cells lysed as estimated by visual inspection. After centrifugation and filtration, the crude cell extract showed 12.47±2.31 mg/ml, as measured by NanoDrop at 280 nm. The total volume of the cell extract was around 60 ml.
@@ Line 184: / 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;width:700px;">
+   <tr>
+    <td style="border:none;"><img src="https://static.igem.org/mediawiki/2015/5/50/Uppsala_sds_1.png"></td>
+    <td style="border:none;"><img src="https://static.igem.org/mediawiki/2015/9/99/Uppsala_sds_2.png"></td>
+   </tr>
+  </table>
+  <figcaption><b>Figure 9.4</b>: SDS-PAGE results of the IMAC fractions of the modified laccase (left) and CueO (right)</figcaption>
    <img src="https://static.igem.org/mediawiki/2015/2/29/Uppsala_figur1grp1123123.png" style="width:350px">
-   <figcaption><b>Figure 9.4: shows 9 fractions from Ion exchange column; one band of 20-30kDa.</b> </figcaption>
+   <figcaption><b>Figure 9.5</b>: shows 9 fractions from Ion exchange column; one band of 20-30kDa. </figcaption>
    <u><b><p>NahR characterization</p></b></u>
    <p>
@@ Line 212: / Line 222: @@
    <u><b><p>IMAC</p></b></u>
     <p>
-    The chromatograms of ModLac and CueO (Ecol) showed very low protein concentrations (see Figures 9.1 and 9.2). The eluate fractions from the IMAC did not show any enzyme activity. Examination of these fractions with SDS-PAGE (see Figure Q) also showed that there were no, or very low concentrations of protein in them. This raises the question as to what went wrong in the purification method. The theory of IMAC rests upon the fact that his-tagged proteins will bind to the column and not elute until a high concentration of imidazole is pumped through the column. Enzyme activity was found in the lysate of CueO and its mutant, which means that there were functioning proteins prior to the IMAC. Possible explanations as to why no activity was measured in the eluate could be that the protein concentration was too low in the elution fraction (due to dilution) or that no protein bound to the column. This was confirmed when enzyme activity was found in the initial wash of the column. These results point toward the fact that something went wrong with the interaction between the protein and the column. It is unlikely that the nickel ions had not bound to the gel, since the columns had a clear light turquoise colour after being loaded. Also, the colour was not washed away during the IMAC procedure. There is a risk that the polyhistidine-tag, that was fused to the proteins, had folded into the protein and was not exposed on the protein surface. The tag needs to be on the surface of the protein to enable interaction with the nickel-enriched gel. This could have been checked by denaturing the protein samples prior to the IMAC, which would ensure that the his-tag was fully unfolded. This would require renaturation of the protein to be able to measure enzyme activity however.
+    The chromatograms of ModLac and CueO (Ecol) showed very low protein concentrations (see Figures 9.1 and 9.2). The eluate fractions from the IMAC did not show any enzyme activity. Examination of these fractions with SDS-PAGE (see Figure 9.4) also showed that there were no, or very low concentrations of protein in them. This raises the question as to what went wrong in the purification method. The theory of IMAC rests upon the fact that his-tagged proteins will bind to the column and not elute until a high concentration of imidazole is pumped through the column. Enzyme activity was found in the lysate of CueO and its mutant, which means that there were functioning proteins prior to the IMAC. Possible explanations as to why no activity was measured in the eluate could be that the protein concentration was too low in the elution fraction (due to dilution) or that no protein bound to the column. This was confirmed when enzyme activity was found in the initial wash of the column. These results point toward the fact that something went wrong with the interaction between the protein and the column. It is unlikely that the nickel ions had not bound to the gel, since the columns had a clear light turquoise colour after being loaded. Also, the colour was not washed away during the IMAC procedure. There is a risk that the polyhistidine-tag, that was fused to the proteins, had folded into the protein and was not exposed on the protein surface. The tag needs to be on the surface of the protein to enable interaction with the nickel-enriched gel. This could have been checked by denaturing the protein samples prior to the IMAC, which would ensure that the his-tag was fully unfolded. This would require renaturation of the protein to be able to measure enzyme activity however.
     </p>
    <u><b><p>ABTS assay on ModLac and CueO</p></b></u>
@@ Line 226: / Line 236: @@
    </p>
    <p>
-   The SDS-PAGE (Figure 9.4) showed that we did not manage to purify the CueO D439A/M510L mutant, though the NanoDrop results shows that there should be 11 g/ml of the protein.  The SDS-PAGE and Nanodrop relative to each other we can establish that the NanoDrop results are unreliable.
+   The SDS-PAGE (Figure 9.5) showed that we did not manage to purify the CueO D439A/M510L mutant, though the NanoDrop results shows that there should be 11 g/ml of the protein.  The SDS-PAGE and Nanodrop relative to each other we can establish that the NanoDrop results are unreliable.
    </p>
    <p>
@@ Line 239: / Line 249: @@
    </p>
    <p>
-   The probable source of error is the buffer. The SDS-PAGE (Figure LOL IEC)results showed that it can be carbonic anhydrase (Can), that is produced during stress and starvation in the BL21DE3 strain and has a pI of 6.4. The overnight had been in the cold room for some time and had started to show floating webs of colonies.
+   The probable source of error is the buffer. The SDS-PAGE (Figure 9.5)results showed that it can be carbonic anhydrase (Can), that is produced during stress and starvation in the BL21DE3 strain and has a pI of 6.4. The overnight had been in the cold room for some time and had started to show floating webs of colonies.
    </p>
@@ Line 335: / Line 345: @@
     </tr>
     <tr>
-     <td>BBa_K1688002</td>
+     <td><a HREF="http://parts.igem.org/Part:BBa_K1688001">BBa_K1688002</a></td>
      <td>RBS + <i>Rhl</i> A</td>
      <td>EcoRI, PstI</td>
@@ Line 352: / 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 BBa_K1688000, well 3: cut BBa_K1688002  and well 4: cut BBa_K1688003. 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 BBa_K1688001 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 BBa_K1688000 + BBa_1688004 (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 443: / 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, BBa_K1688000 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>
+   <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>
    <table>
@@ Line 472: / Line 482: @@
    </tr>
    <tr>
-    <td>BBa_K1688000 in BL21DE3</td>
+    <td><a HREF="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3</td>
     <td>1,0</td>
     <td>2,2</td>
@@ Line 482: / Line 492: @@
    </tr>
    <tr>
-    <td>BBa_K1688000 in DH5α</td>
+    <td><a HREF="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in DH5α</td>
     <td>1,0</td>
     <td>1,6</td>
@@ Line 514: / 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 BBa_K1688000 respectively untransformed and supernatant extracted from <i>E.coli</i> DH5α with BBa_K1688000 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>
-   Table 2 shows that a higher concentration of mono-rhamnolipids causes the drop to expand more and collapse faster. This verifies that presence of rhamnolipids can be indicated from drop collapse tests. The drop from sample BBa_K1688000 in BL21DE3 from table 3 collapsed after 30 seconds and expansion of drop diameter was 120% within 5 minutes from 1 cm to 2.2 cm which indicate presence of biosurfactant. The drop from sample BBa_K1688000 in DH5α collapsed and diameter expansion of drop was 90% after 20 minutes. This indicates some presence of biosurfactants. As expected the test indicate that BBa_K1688000 has higher expression rates and rhamnolipid production was higher in BL21DE3 than in DH5α as BL21DE3 is good for protein expression. The negative controls, LB medium and untransformed BL21DE3 and DH5α showed very little expansion or no expansion, which is expected as they do not produce biosurfactants.
+   Table 2 shows that a higher concentration of mono-rhamnolipids causes the drop to expand more and collapse faster. This verifies that presence of rhamnolipids can be indicated from drop collapse tests. The drop from sample <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> in BL21DE3 from table 3 collapsed after 30 seconds and expansion of drop diameter was 120% within 5 minutes from 1 cm to 2.2 cm which indicate presence of biosurfactant. The drop from sample <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> in DH5α collapsed and diameter expansion of drop was 90% after 20 minutes. This indicates some presence of biosurfactants. As expected the test indicate that <a href="http://parts.igem.org/Part:BBa_K1688000"><span class="res_link">BBa_K1688000</span></a> has higher expression rates and rhamnolipid production was higher in BL21DE3 than in DH5α as BL21DE3 is good for protein expression. The negative controls, LB medium and untransformed BL21DE3 and DH5α showed very little expansion or no expansion, which is expected as they do not produce biosurfactants.
    </p>
    <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  BBa_K1688000 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, BBa_K1688000 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 550: / Line 560: @@
     <tr>
      <td>2</td>
-     <td>BBa_K1688000 in BL21DE3</td>
+     <td><a HREF="http://parts.igem.org/Part:BBa_K1688000">BBa_K1688000</a> in BL21DE3</td>
      <td>10,1</td>
      <td>12,3</td>
@@ Line 585: / 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 BBa_K1688000 and standard mono-rhamnolipid 10, 30 respectively 50 μg. The detection spot of BBa_K1688000 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 BBa_K1688000 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 (BBa_K1688000). 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 (BBa_K1688000). 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 (BBa_K1688000).</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 BBa_K1688000. 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 (BBa_K1688000) 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.
+   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>