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	<p align="justify"><i>E. coli</i> BL21 were transformed with the operon and grown to a OD of 0.6. A negative sample was taken before IPTG was added to a concentration of 1mM for induction. Cells stayed at 28°C for 12 hours and later were harvested and resuspended in buffer. A small amount of both induced samples and negative samples was loaded on a SDS-PAGE while proteins were extracted from the rest. The SDS-PAGE showed overexpression of proteins of the expected mass.		<p align="justify"><i>E. coli</i> BL21 were transformed with the operon and grown to a OD of 0.6. A negative sample was taken before IPTG was added to a concentration of 1mM for induction. Cells stayed at 28°C for 12 hours and later were harvested and resuspended in buffer. A small amount of both induced samples and negative samples was loaded on a SDS-PAGE while proteins were extracted from the rest. The SDS-PAGE showed overexpression of proteins of the expected mass.
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−	Our cells were again inoculated and induced at an OD of 0.6. This time we added <small>D</small>-xylose at an concentration of 4g/l. After induction for 12 hours cells were harvested and lysated. The cell lysate was chemically extracted with dichlormethan and analysed with HPLC-MS. Unfortunately in our measurement no ethylene glycol could be verified. It is possible that overexpression of the other enzymes of the pathway is necessary for production in <i>E. coli</i>.	+	Our cells were again inoculated and induced at an OD of 0.6. This time we added <small>D</small>-xylose at an concentration of 4g/l. After induction for 12 hours cells were harvested and lysated. The cell lysate was chemically extracted with dichlormethan and analysed with HPLC-MS. Unfortunately in our measurement no ethylene glycol could be verified. It is possible that overexpression of the other enzymes of the pathway is necessary for production in <i>E. coli</i>.</p>
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Revision as of 02:43, 19 September 2015

D-xylonic acid producing operon

D-Xylose is a monosaccharide belonging to the aldopentose family. It was recently shown that the D-xylose dehydrogenase xylB from Caulobacter crescentus can convert D-xylose to D-xylonolactone. This can react spontaneously or through the catalysation of xylC to D-xylonic acid. (2)

In E. coli D-xylonic acid can further be metabolized to ethyleneglycol by the enzymes yjhG (BBa_K1602012), yagE (BBa_K1602011) and yqhD (BBa_K1602013) which are already present in this host. (1)

Usage

This part is a composite of two coding genes with strong RBS (BBa_B0034). The transcription is controlled by a T7 promotor (BBa_I719005).

We used this operon to investigate possible production of ethylene glycol in E. coli.

T7-promotor (BBa_I719005) ribosome binding site B0034 (BBa_B0034) D-xylose dehydrogenase xylB (BBa_K1602009) D-xylonolactone lactonase xylC (BBa_K1602010)

Results

E. coli BL21 were transformed with the operon and grown to a OD of 0.6. A negative sample was taken before IPTG was added to a concentration of 1mM for induction. Cells stayed at 28°C for 12 hours and later were harvested and resuspended in buffer. A small amount of both induced samples and negative samples was loaded on a SDS-PAGE while proteins were extracted from the rest. The SDS-PAGE showed overexpression of proteins of the expected mass.
Our cells were again inoculated and induced at an OD of 0.6. This time we added D-xylose at an concentration of 4g/l. After induction for 12 hours cells were harvested and lysated. The cell lysate was chemically extracted with dichlormethan and analysed with HPLC-MS. Unfortunately in our measurement no ethylene glycol could be verified. It is possible that overexpression of the other enzymes of the pathway is necessary for production in E. coli.

Figure 2 Scan of the PAGE containing four different samples: marker (M; Protein Marker III AppliChem); samples 1-4 reference and induced.	Figure 3 Plot of the gel lanes based on contrast analyses - created with ImageJ
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Figure 4: HPLC-MS spectra from cell lysate with artificialy added ethylene glycol
Figure 5: Our culture which was induced with xylose showed no ethyleneglycol after extraction in mass spectrometrie