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Revision as of 20:35, 15 September 2015

Results

Enzymatic degradation

Naphthalene pathway

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.

Verification of transcription of genesrhlA and rhlB with dTomato as reporter

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 was more expressed and rhamnolipid production was higher in BL21DE3 than in DH5α as BL21DE3 is good for protein expression. The negative controls, LB medium and un-transformed BL21DE3 and DH5a 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 un-transformed, 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 un-transformed 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.

Conclusion

Although our tests, drop collapse test, CTAB test and TLC, indicated the presence of biosurfactants, rhamnolipids, and mono-rhamnolipids respectively, we still have to perform quantitative assays such as mass spectrometry, HPLC, etc. to identify the concentration of rhamnolipids produced. The data that could be obtained from these assays will help in maintaining the bacterial growth in the degradation reactor. 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.