Results

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• Enzymatic degradation
• Naphthalene pathway
• Biosurfactants

Enzymatic degradation

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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.

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.

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.

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.

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.

Results of the enzymatic assay measurements are shown on Figure 5. Determined values were Vmax=32.64 µM.min^-1, and K_m=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.

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.)

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.

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.

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).

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.

Catechol 1,2 dioxygenase was successfully expressed and extracted in large quantities, while preserving its activity, hence proving E.coli as a potent host for its production. Since a major point of the project is to export the degrading enzymes out of the cell, the primary focus was on pH and temperature stability, as well as Km to find its optimal working conditions. The dioxygenase showed good stability in a wide temperature range, losing its activity completely only in extreme conditions, as displayed in figure 6. The enzyme had its highest residual catalytic functions at temperatures around 40 °C, which is beneficial for our purposes to create a bioreactor since the enzyme works in the same temperatures as the cells will grow. It’s pH range was narrow, showing a peak around pH 7, see figure 7. These results, showing that the enzyme will be able to work optimally in the same pH as our cells, was also seen as good results considering our bioreactor. Dioxygenase showed very high substrate specificity and quickly approached Vmax with increase in catechol concentrations, shown in figure 5. This is favorable for extracellular conditions, where any substrate produced by laccases is diluted several orders of magnitude.

BPB-Test

The bromophenol blue plate test with the CueO and CotA showed clear regions of a brighter colour in the agar around the cells that contained PAH-degrading enzymes, thus this indicated the presence of laccases. There was no significant difference between the CueO with or without the HlyA-tag. On our negative controls, less bromophenol blue had been degraded than on the parts on the plate containing CueO and CotA. This indicated that it actually was our enzymes that were responsible for breaking down the bromophenol (see figure 8). The results of the bromophenol test with liquid cultures was however hard to assess, since we did not have any positive controls to compare with.

NahR/Psal

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.

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.

ABTS assay on ModLac and CueO

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.

Ion-exchange chromatography

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 results clearly showed that it’s not CueO D439A/M510L mutant in the SDS-PAGE. The NanoDrop results can be discarded since 11g/mL also it not an accurate result of the protein concentration, by itself and compared to the SDS-PAGE results.

The error sources could be many but when going through the method of choice; there is an obvious error in the pH of the buffer. The theoretical isoelectric point of CueO D439A/M510L mutant is 6.5 and the buffers pH differs med +0.2. This is not a sufficient pH for making the CueO D439A/M510L mutant negatively charged. This however may not only be the only source of error. No protease inhibitor was used during the experiment, thus the proteins could also have been degraded.

The IEC is an anion exchange column and was used earlier with good results. It was also thoroughly washed and equilibrated with the mixed buffer.

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.

Naphthalene pathway

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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.

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.

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

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Gel electrophoresis

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

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

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.

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

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

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

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

TLC

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 10-12 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.