Part:BBa_K1497015:Design
Pelargonidin producing operon - T7-B0034-F3H-B0034-DFR-B0034-eANS
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 239
Illegal BamHI site found at 729
Illegal BamHI site found at 1535 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1287
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1272
Design Notes
To analyze the pelagonidin production operon (K1497015), we transformed it into E.coli Bl21(DE3). An overnight LB culture was used to inoculate an expression-culture. The expression of pelargonidin was performed according to Yan et al., (2007). After the induction with 1 mM Isopropyl-β-D-thiogalactopyranosid (IPTG) E.coli BL21 (DE3) cells were transferred into M9-media and fermented for 48h at 37°C in present of 0.1 mM naringenin.
Figure 2 E.coli BL21 (DE3) pellet containing the pelargonidin producing operon after the fermentation. According to Yan et al. (2007) a pelargonidin producing E.coli should be red after a pelargenidin production. The operon with the engineered anthocyanindin synthase produces more pelargonidin |
After the expression of pelagonidin producing operon with engineered ANS (K1497015) in present of 0.5 mM narigenin we performed an extraction of pelargonidin with methanol /dichloromethane from the pellet and supernatant and verified the pH-dependency of pelargonidin (Fig. 3).
Figure 3 Extracted pelargonidin from E.coli BL21 (DE3) under day light. The color of pelargonidin depends on pH value and solvent. This indicates the present of pelargonadin. Left: Methanol extraction; right: Dichlormethane extraction. |