Team:Uppsala/Results

Results

Enzymatic degradation

Restriction free cloning (RFC)

The first PCR-step in the RFC was run with different concentrations of DMSO to inquire into what concentration would be optimal. The results of this PCR is shown in Figure 1.

Figure 1: Agarose gel with the PCR products from the first test PCR with different DMSO concentrations. The first 4 wells in the upper left corner has 0% DMSO, the 4 wells in the upper right corner has 3% DMSO, the 4 wells in the lower left corner has 5% DMSO and the last 4 wells has 10% DMSO. The upper bands are the PCR products and the lower are primer dimers. The gel shows that the PCR products are 250 bp long, which is the correct length. The products with 5% DMSO have the sharpest bands on the gel.

The results from the previous PCR showed that 5% DMSO was the optimal concentration for our primers and therefore a new PCR was run under the same conditions as previously: This was to yield more product that could be used in the next step of the PCR. The results from this PCR is shown in Figure 2.

Figure 2: 2% agarose gel with PCR product (primers) from the first PCR done with 5% DMSO. Wells 11 to 20 contain primers for catechol 1,2 dioxygenase and CueO. These have the correct length; around 250 bp. The other wells contain negative controls. The upper bands show primer dimers.

After the second PCR the PCR products (pSB1C3 backbones with the laccases CotA and CoeO with inserted HlyA-tag as well as catechol 1,2 dioxygenase with inserted HlyA-tag) were transformed into competent DH5a after digestion of methylated plasmids with Dpn1. After incubation of the transformed cells on agar plates with chloramphenicol overnight, colonies had appeared on the plates with dioxygenase and CueO. The RFC did however not seem to work for the CotA. The colonies were screened using colony PCR and the result from this PCR is shown in Figure 3. The results shown in this figure indicates that the RFC was successful for both the catechol 1,2 dioxygenase and the CueO.

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.

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.

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.

Figure 4. SDS PAGE of elution fractions. 20 µl of each fraction (and BSA stock), and 10 µl ladder were run on 10% acrylamide gel to confirm protein presence in the fraction. L: PageRuler Unstained Protein Ladder; B: BSA, 200µg/ml; C: crude cell extract; 0-1000: Different fractions as indicated of millimoles of NaCl present. Image is composite of two gel pictures and is digitally enhanced for contrast and clarity with demonstrative purpose.

Collected fractions of each salt concentration (0 to 500 mM NaCl in 50 mM steps) were then ran on an SDS PAGE gel to confirm presence of the catechol 1,2 dioxygenase enzyme. The results, shown in Figure 4, confirmed that the protein is being successfully expressed in large quantities and is of the expected size. The peak of the protein was spread between the 300 mM and 350 mM step. Each fraction was then measured for protein concentration and the results for 300 and 350 fractions were 879 µg/ml, and 1161 µg/ml respectively.

Figure 5Initial velocity versus substrate concentration plot. Vmax = 32,64 µM/min, and Km = 16,85 µM-1. Individual data points not shown.
Figure 6Enzymatic activity depending on temperature. Catechol exhibits highest activity at 40 °C, making 50,625 µM of product in 1 minute, and very low amounts of product at temperatures above 55 °C or below 15 °C.

Results of the enzymatic assay measurements are shown on Figure 5. Determined values were Vmax=32.64 µM.min-1, and Km=16.85 µM-1. The enzyme showed highest velocity at 150µM concentration of catechol, but for the rest of the tests, 100 µM was used instead. The enzyme exhibits strong substrate inhibition above 400 µM substrate concentration, with 10mM completely inhibiting enzyme activity (data not shown in Figure 5). Graphical representation of the results from the measurement of activity depending on temperature is shown in Figure 6. Results obtained from the assay of enzyme activity at different pH are presented on Figure 7.

Figure 7: pH optimum of C12D. The enzyme shows a pH optimum in the neutral range, with slight bias towards alkaline pH.

Maximum velocity of 34,7875 µM.min-1 was obtained ¨at pH 8,16 and 22°C, with very similar results for neutral pH as well (33,9 µM.min-1 at pH 7,17). pH lower than 5 completely inhibits enzyme activity, as well as pH>9.

Bromophenol blue test

A composite picture of 4 simultaneous bromophenol blue tests is shown on Figure 8. A slight difference in color can be seen between the negative control (1) with dioxygenase and the colonies with laccases (CotA (3) and CueO (4)). This indicates that the laccases are able to degrade the bromophenol blue which is a positive result. (When performing this experiment the frameshift in the HlyA-tag had not yet been discovered and therefore the result in section number 2 is not relevant.)

Figure 8. Bromophenol blue assay. This composite image contains 4 separate plates, restreaked with a single colony of dioxygenase with J23110 promoter (1, negative control), CueO with BBa_J23110 promoter and Hlya tag (2) CotA with BBa_J23110 promoter (3) and CueO with J23110 promoter (4)

Removal of the HlyA-tag from ModLac

To ensure that the fused HlyA tag did not affect the enzymatic activity of the laccase, the HlyA tag was removed before characterization of the modified laccase. This was done by digesting with the restriction enzyme Kpn1, ligating it together and PCR amplifying the segments with VF2 and VR primers. This resulted in two main segments: the ModLac without tag and the ModLac re-ligated with the HlyA tag. To separate these two segments, the PCR product was run on an agarose gel, and the shorter segment was extracted from the gel.

Figure 9:

Purification of ModLac and CueO (Ecol)

The resulting chromatograms of the IMAC purifications of ModLac and CueO (Ecol) can be seen below in Figure 9.1 and 9.2.

Figure 9.1: Chromatograms of all IMAC fractions of ModLac. All fractions can be seen in the left chromatogram while the last 15 fractions can be seen in the right. A big peak in absorbance is visible around fraction No. 2 while only small peaks can be seen in the left picture.
Figure 9.2: Chromatograms of all IMAC fractions of CueO(Ecol). All fractions can be seen in the left chromatogram while the last 15 fractions can be seen in the right. A big peak in absorbance is visible around fraction No. 2 while only minor peaks can be seen in the left picture.

ModLac characterization

The breakdown of ABTS was measured at 420 nm using a spectrophotometer. The stable oxidized form of ABTS absorbs at 420 nm. ModLac laccase was concluded to have a higher enzymatic activity than the CueO laccase.

NahR characterization

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).

Figure 10:NahR/Psal induction test in liquid culture. Expression of dTomato under the NahR/Psal promoter system with different salicylate concentrations.
Figure 11: NahR/Psal induction test on agar plate. Concentration of salicylate from left to right: 0,1 μM, 1 μM, 10 μM, 100 μM and 1 mM.

Conclusions

The result of the first test PCR on dioxygenase, CotA and CueO, displayed in figure 1, showed that a 5% concentration of DMSO gave the best results for this PCR. This could be seen on the bands on the gel, which were thicker when 5% DMSO had been used. The results displayed in figure 2 showed that the part to be inserted had the correct length. This indicated that the insert contained the right gene with the overhangs, which is important for the next PCR to be successful. When the RFC products were transformed into the cells the fact that there were colonies on the plates confirmed that the RFC was successful. The cells would not have survived if they did not have a plasmid with chloramphenicol resistance because they were grown on agar plates with chloramphenicol. Because the original plasmids are methylated from been amplified in E.coli cells they can be degraded with DpnI and theoretically the only plasmids that are left should be our wanted plasmids. This means that the PCR product from the first PCR containing the HlyA-tag worked successfully as primers in the second PCR and hence since the HlyA-tag is the main part of the primer, it should have been inserted. According to the results in figure 3 the HlyA-tag was successfully attached to the dioxygenase and CueO since the RFC products were around 200 bp longer than the original genes.The RFC products did however encode defective export tags due to faulty primers. No extensive experiments to test the effect of the faulty enzyme were done.

Naphthalene pathway

Lifting of naphthalene pathway

Our concrete goals in the lab were to extract the naphthalene degrading pathway with genes NahA to NahF both with and without its native promoter through PCR. As is confirmed by colony PCR and electrophoresis, the pathway was successfully lifted from the Nah7 plasmid.

Assembly of promoter, pathway and backbone

The PCR product, namely the naphthalene pathway, was successfully assembled into a standard iGEM backbone through 3A assembly, confirmed through colony PCR, and two different promoters were added through standard assembly due to unwanted restriction sites in the pathway sequence.

Sequencing and proteomics

Due to financial and temporal limitations, no sequencing or proteomic studies could be performed.

Plates with naphthalene in lid

To assess differences in survivability between the cells containing the naphthalene degrading pathway and negative control cells containing an RFP-coding gene. Plates were split in two parts, one containing the naphthalene degrading bacteria and one with the negative control. Fixed amounts of naphthalene crystals ranging from 50 mg to 2 g were then placed in the lid of each plate, to determine the difference in growth rate. However, no visible difference was observed. These results are consistent with results from experiments with liquid cultures where naphthalene was also supplied in gas form.

Figure 4: shows the survivability rates of modified DH5α E.coli in comparison to the negative control at a different naphthalene concentrations. Increasing concentrations from left to right. Modified DH5α are to the left of each plate, negative control on the right.

Cultures with naphthalene

The upper naphthalene pathway was introduced into both DH5α and BL21 strains of E.coli, 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.

Both strains were grown in liquid culture with either no naphthalene, naphthalene directly supplied to the medium, or with naphthalene supplied in gas form as shown in figure 5. The OD of the cultures were measured after 24 and 48 hours at both OD600 (for cell growth) and for OD303 (for the presence of salicylate). The graphs in figures 6 to 11 show the experimental values obtained by spectrometry.

Figure 5 shows liquid cultures grown under three different conditions. From left to right: 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 last picture shows a liquid culture with naphthalene dissolved directly into the medium from below.
Figure 6 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.
Figure 7 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.
Figure 8 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.
Figure 9 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.
Figure 10 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.
Figure 11 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.

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 naphthalene supplied in gas form did not appear to affect the cell growth at all. However, in cultures without naphthalene the negative control grew somewhat better than cells with the construct. The reason is probably that the negative control did not have to maintain a large unnecessary plasmid.

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. Levels of salicylate, however, appear to be far higher in cultures without naphthalene or with naphthalene in gas form, disagreeing with our original hypothesis. This may be due to interfering cells or substances. Regardless, results still show a clear trend both in salicylate levels and in cell growth, indicating that our construct is indeed being expressed and is degrading naphthalene to salicylate.

Biosurfactants

Gel electrophoresis
Table 1: Biobricks used for gel electrophoresis, their inserts, restrictions enzymes used for digestion, lengths of inserts and plasmid backbones and expected band lengths.
Biobrick Code Insert Digestion Insert (bp) Backbone pSB1C3 (bp) Expected bands
BBa_K1688000 Promoter + RBS + Rhl A + RBS + Rhl B EcoRI, PstI 2333 2070 2374, 2037
BBa_K1688001 RBS + Rhl A + RBS + Rhl B XbaI, PstI 2333 2070 2324, 2052
BBa_K1688002 RBS + Rhl A EcoRI, PstI 2298 2070 1006, 2037
BBa_K1688003 RBS + Rhl B EcoRI, PstI 1325 2070 1366, 2037
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.
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.

Figures 3 and 4 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
Figure 5: E.coli DH5α transformed with assembled product BBa_K1688000 + BBa_1688004 (dTomato construct) on agar plate.

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.

Table 2: Data from drop collapse test for different concentrations of standard mono-rhamnolipids. Diameter of drop after 0, 5, 10, 15 and 20 min, expansion of drop diameter in percentage and if the drop collapsed.
Standard mono-rhamnolipids mg/ml Diameter of drop (cm) at different time intervals Expansion pf drop % Collapse
0 min 5 min 10 min 15 min 20 min
0 - control 0,65 0,65 0,65 0,65 0,65 0% No
0,2 0,75 0,9 0,9 0,9 0,9 20% No
0,4 0,75 0,95 0,95 0,95 0,95 27% No
0,6 0,75 1 1 1 1 33% After 1 min
1 0,75 1,2 1,2 1,2 1,2 60% Collapse immediately within 30 seconds
1,6 0,8 1,65 1,8 1,8 2,2 187% Collapse immediately within 30 seconds
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
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.
Sample (50 µl) Diameter of drop (cm) at different time intervals Expansion pf drop % Collapse
0 min 5 min 10 min 15 min 20 min
LB 0,65 0,65 0,65 0,65 0,65 0% No
BBa_K1688000 in BL21DE3 1,0 2,2 2,2 2,2 2,2 120% After 0:30 min
BBa_K1688000 in DH5α 1,0 1,6 1,75 1,75 1,9 90% After 1:00 min
BL21DE3 0,75 0,75 0,9 1,0 1,0 33% No
DH5α 0,8 0,8 0,8 0,8 0,8 0% No
Figure 7: A bar graph displaying the expansion of drop of different samples. Data from table 3

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 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.

CTAB
Figure 8: in E.coli BL21DE3 cells with BBa_K1688000 on CTAB plate.

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

TLC
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.
Table 4: Retention factor (Rf) of different samples run on TLC silica plate.
Lane Sample Distance moved by sample (cm) Distance moved by solvent (cm) Rf value
1 BL21DE3 No spot 12,3 -
2 BBa_K1688000 in BL21 10,1 12,3 0,82
3 P.putida No spot 12,3 -
4 Standard mono-rhamnolipids (10 mg/ml) 1μl 10,3 12,3 0,83
5 Standard mono-rhamnolipids (10 mg/ml) 3 μl 10,2 12,3 0,82
6 Standard mono-rhamnolipids (10 mg/ml) 5 μl 10,2 12,3 0,82

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. 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
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.
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.
Figure 12: The mass spectrum from total ion chromatogram (TIC) of lipid extraction of E.coli BL21DE3 with RhlA and RhlB gene (BBa_K1688000).

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.

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.