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−	<div><b>Figure 2</b> <p align="justify">Scan of the PAGE containing four different samples from left to right 1 - induced, 1 - uninduced, 2 - induced, 2 - uninduced, 3 - induced, 3 - uninduced, 4 - induced, 4 - uninduced. At the right there are two rows of marker (M; Protein Marker III AppliChem).</p></div>	+	<div><b>Figure 2</b> <p align="justify">Scan of the PAGE containing four different samples: marker (M; Protein Marker III AppliChem); samples 1-4 reference and induced.</p></div>
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Revision as of 00:41, 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.

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.

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