Team:Uppsala/Results

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.
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, BL21 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.
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 BL21 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 BL21 than in DH5α as BL21 is good for protein expression. The negative controls, LB medium and un-transformed BL21 and DH5a showed very little expansion or no expansion, which is expected as they do not produce biosurfactants.