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

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

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

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

   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.  

 

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   The regulative capabilities of the NahR promoter system was tested under different salicylate concentrations. dTomato was attached after the NahR biobrick (BBa_J61051) http://parts.igem.org/Part:BBa_J61051 as a reporter gene, and the fluorescence was measured using our fluorometer prototype. (Link to fluorometer).  

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   We initially tested the NahR/Psal promoter system with dTomato on a pSB1C3 plasmid. The cells containing the plasmid were streaked on agar plates with different salicylate concentrations in the agar medium. Similarly, we also examined liquid cultures with different concentrations of salicylate in the solution. Both of these tests showed that the bacteria were producing the dTomato protein in presence of salicylate, independent of the salicylate being in liquid culture or in the solid growth medium. It can also be easily observed that the fluorescence increased with increasing salicylate concentrations (See figure 10 and 11). The measurements were made with  the fluorometer that our team built in order to be able to characterize this system. These results were compared to MACS measurements on the same colonies, and can be viewed on the fluorometer site. This is a further improvement of the  characterization of BBa_J61051 done by the iGEM team Peking 2013.

   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.   

   ABTS is a commonly used substrate when evaluating reaction kinetics of specific enzymes. Due to its reduction potential, it acts as an effective electron donor. Since we are working with laccases, which are multi copper oxidases, which oxidize substrates, ABTS is a suitable substrate. ABTS will donate electron to reduce molecular oxygen. The oxidized ABTS has a different absorption spectrum and the reaction can thus be observed in a spectrophotometer.

   The enzyme assays of the lysates with ABTS showed a significant difference in enzyme activity between CueO and the mutant CueO, also known as ModLac (see Figure 9). In an article by Kataoka’s research group, the investigated CueO mutant showed to have a higher enzyme activity than the wild type (Kataoka K et al. 2012). This is confirmed by our results. The ModLac is designed so that the active site is the same as the double mutant that Kataoka and his research team investigated, but with some modifications at the C- and N-terminus. These modifications were made so that the enzyme would be exported from the cell and so that it could be easily purified. Restriction sites were added around these features so that it could be easily manipulated by our team and other iGEM teams that would like to use it for different purposes. These modifications could have affected the enzymatic activity by indirectly interfering with the active site. We tried to prevent this by adding a flexible linker sequence between the sequence coding for the laccase and the HlyA-export tag so that it would fold correctly. The conclusion that can be made after observing the results is that the designed ModLac have a better enzymatic activity than the wild type Laccase CueO (BBa_K863006). We have therefore improved the function of an already existing biobrick and supplemented further characterization of wild type CueO (BBa_K863006) when it is compared with ModLac. The new biobrick that we have designed has additional properties that makes it easier to manipulate than the wild type with simple tools in biotechnology.  

   During the purification of the modified laccase, we tried using an ion-exchange column since the IMAC results was unsatisfying. However, to verify which fraction contained the CueO D439A/M510L mutant we used the Nanodrop and SDS-PAGE. The SDS-PAGE showed that the fractions didn’t contain any protein of significance (Shown in Figure LOL) and the NanoDrop showed some clearly irregular numbers. Spectrophotometric measurements of the fractions with ABTS also confirmed what the SDS-PAGE showed, there seemed to be no proteins in the fractions, or they were very diluted (Data not shown).

   The SDS-PAGE (see fig LOL.) 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 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 BL21-DE3 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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   The upper naphthalene pathway was introduced into both DH5α and BL21 strains of <i>E.coli</i>, where DH5α is a cloning strain and BL21 is a strain optimized for protein expression. In the DH5α cells a medium strength promoter was used to put less strain on the cells, whereas in BL21 a strong promoter could be used to increase the expression level of the desired enzymes.

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   <figcaption><b>Figure 6</b> shows the average OD₆₀₀ values of two different experiments, after the cells had been grown for 24 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.</figcaption>

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   <figcaption><b>Figure 7</b> shows the OD₆₀₀ value for one experiment, after the cells have been grown for 48 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.</figcaption>

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   <figcaption><b>Figure 8</b> shows the average OD₃₀₃ values of two different experiments, after the cells have been grown for 24 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.</figcaption>

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   <figcaption><b>Figure 9</b> shows the average OD₃₀₃ value of one experiment, after the cells have been grown for 48 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.</figcaption>

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   <figcaption><b>Figure 10</b> shows the average OD₃₀₃ values of the supernatant from two different experiments, after the cells have been grown for 24 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.</figcaption>

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   <figcaption><b>Figure 11</b> shows the average OD₃₀₃ value of one experiment, after the cells have been grown for 48 hours. The cells were grown under three different conditions; without naphthalene, with 500 mg of naphthalene dissolved directly into the medium and with 500 mg of naphthalene in an eppendorf tube suspended above the culture. The values displayed are correspondent to constructs: DH5α-pSB3C5-Upper naphthalene pathway with promoter BBa_J23101, BL21-pSB3C5-Upper naphthalene pathway with promoter BBa_J23110 and DH5α-pSB1C3-RFP insert.</figcaption>

   All the cultures containing naphthalene supplied directly to the medium showed a clear trend of significantly lower growth rates in the negative control compared to the cells containing the pathway. This is to be expected as naphthalene is toxic to the cells and the negative control is unable to degrade it. After 24 hours, the BL21 had grown substantially more than both the negative control and the DH5α cells. This is not surprising as this strain is better at producing the enzymes of our pathway, and thus should be better at degrading naphthalene. However, after 48 hours the DH5α culture had reached similar levels of optical density as the stabilized BL21 cultures. A plausible reason is that the strain still has the pathway, though the level of expression is lower than in BL21.  

   The presence of salicylate both directly in the culture and with the cells removed, was measured at OD₃₀₃. Similar results were observed at 24 hours as in above described experiment, with higher salicylate levels in BL21 compared to DH5α and the negative control. After 48 hours DH5α had approximately as high levels of salicylate as BL21.  

     <td>BBa_K1688000 </td>

     <td>BBa_K1688001</td>

     <td>BBa_K1688002</td>

     <td>BBa_K1688003</td>

   <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 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>

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

   <figcaption><b>Figure 5</b>: <i>E.coli</i> DH5α transformed with assembled product BBa_K1688000 + BBa_1688004 (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 <i>rhlA</i> and <i>rhlB</i> are transcribed.

   <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>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>

   <td>BBa_K1688000 in BL21DE3</td>

   <td>BBa_K1688000 in DH5α</td>

   <figcaption><b>Figure 7</b>: 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 <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 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 BL21 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.

   <figcaption><b>Figure 8</b>: in <i>E.coli</i> BL21DE3 cells with  BBa_K1688000 on CTAB plate.</figcaption>

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

   <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>

     <td>BBa_K1688000 in BL21</td>

   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.  

   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.

   <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 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 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>

   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.   

   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.  

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