Difference between revisions of "Part:BBa K1497015"

(→‎Functional Parameters)
Line 48: Line 48:
 
===Functional Parameters===
 
===Functional Parameters===
  
−
To analyze the pelagonidin production operon (<html><a href="/Part:BBa_K1497015">K1497015</a></html>), we transformed it into <i>E.coli</i> 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) <i>E.coli</i> BL21 (DE3) cells were transferred into M9-media and fermented for 48h at 37°C in present of 0.1 mM naringenin.
+
To analyze the pelagonidin production operon (<html><a href="/Part:BBa_K1497015">K1497015</a></html>), we transformed it into <i>E.coli</i> Bl21(DE3). An overnight LB culture was used to inoculate an expression-culture. The expression of pelargonidin was performed according to Yan <i>et al.</i> (2007). After induction with 1 mM Isopropyl-β-D-thiogalactopyranosid (IPTG) <i>E.coli</i> BL21 (DE3) cells were transferred into M9-media and fermented for 48h at 37°C in present of 0.1 mM naringenin.
  
 
<html>
 
<html>

Revision as of 21:25, 17 October 2014

Pelargonidin producing operon - T7-B0034-F3H-B0034-DFR-B0034-eANS




Pelargonidin is an anthocyanidin. Anthocyanidins are the non-glycosylated precursos of anthocyanins, which are water-soluble vacuolar pigments that appear yellow to dark-red (pH-dependent) and which are responsible for color of flowers and fruits and are health-promoting for humans. The iGEM Team TU Darmstadt 2014 constructed a pelargonidin producing operon under the control of a T7 promoter (K1497014 and K1497015, respectively). The operon consists of 3 genes (flavonon-3beta-hydroxylase, dihydroflavonol 4-reductase, anthocyanindin synthase) each with strong RBS (Fig.1) This operon catalysis the reaction from naringenin to pelargonidin (Fig. 2).

The F3H gene from Petroselinum crispum and the DFR gene from Dianthus gratianopolitanus were kindly provided by Dr. Stefan Martens (Research and Innovation Centre, Fondazione Edmund Mach, Italy). The ANS from Fragaria x ananassa was E. coli codon optimized and synthezised by Eurofins MWG Operon.


Figure 1 A: Genetic map of pelargonidin producing operon. R: RBS; F3H: flavonon-3beta-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanindin synthase.B: Reaction scheme of a pelargonidin producing operon

Functional Parameters

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 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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 239
    Illegal BamHI site found at 729
    Illegal BamHI site found at 1535
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1287
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1272


References

1. Yan Y, Chemler J, Huang L, et al. (2005) Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli. 71:3617–3623. doi: 10.1128/AEM.71.7.3617

2. Yan Y, Li Z, Koffas M a G (2008) High-yield anthocyanin biosynthesis in engineered Escherichia coli. Biotechnology and bioengineering 100:126–40. doi: 10.1002/bit.21721