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Revision as of 16:31, 18 September 2015

Results

Enzymatic degradation

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 does 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 does 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. Once again, 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 genesrhlA 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 hag 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 expressing biobrick BBa_K1688000. The m/z 447 ion chromatogram corresponding to (M-H)- of Rha-C8-C8 (mono-rhamnolipid) with retention time at 2.82.

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 PAHs 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.