Difference between revisions of "Part:BBa K1497023:Experience"

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===Applications of BBa_K1497023===
 
===Applications of BBa_K1497023===
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<html>For improving the enzyme the iGEM team TU Darmstadt 2014 decided to remove the tail and to construct their pelargonidin operon with this engineered ANS (eANS). The laboratory results cover the previous modeling results. The engineered ANS exhibited better yields than the original one when used in an operon producing pelargonidin (<a href="/Part:BBa_K1497015">BBa_K1497015</a>). </html>
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To analyze the pelargonidin 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.
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  src="https://static.igem.org/mediawiki/parts/d/d7/Pelletf%C3%A4rbungII.png"></p>
 
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      <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
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<i>E. coli</i> BL21 (DE3) pellet containing the pelargonidin producing operon after the fermentation. According to Yan et al. (2007) a pelargonidin producing <i>E. coli</i> should be red after a pelargonidin production. The operon with the engineered anthocyanidin synthase produces more pelargonidin.</span></p>
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After the expression of pelagonidin producing operon with engineered ANS (<html><a href="/Part:BBa_K1497015">K1497015</a></html>) 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).
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 3</b></span></a><span lang="EN-US">
 
       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 3</b></span></a><span lang="EN-US">
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<i>E.coli</i> BL21 (DE3) pellet containing the pelargonidin producing operon after the fermentation. According to Yan et al. (2007) a pelargonidin producing <i>E.coli</i> should be red after a pelargenidin production. The operon with the engineered anthocyanindin synthase produces more pelargonidin</span></p>
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Extracted pelargonidin from <i>E. coli</i> BL21 (DE3) under day light. The color of pelargonidin depends on pH value and solvent. This indicates the present of pelargonidin. Left: Methanol extraction; right: Dichlormethane extraction.</span></p>
 
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Latest revision as of 00:15, 18 October 2014


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Applications of BBa_K1497023

To analyze the pelargonidin 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 pelargonidin production. The operon with the engineered anthocyanidin 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 pelargonidin. Left: Methanol extraction; right: Dichlormethane extraction.

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