Difference between revisions of "Part:BBa K1602017"
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===<h2>Results</h2>=== | ===<h2>Results</h2>=== | ||
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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.</p> |
− | < | + | <p align="justify">In a NAD<sup>+</sup> assay activity of <i>xylB</i> the activity of <i>xylB</i> could been proven</p> |
− | 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 significant production in <i>E. coli</i>.</p | + | <p align="justify">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 significant production in <i>E. coli</i>.</p> |
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+ | <img class="shrinkToFit transparent" alt="https://static.igem.org/mediawiki/parts/4/4f/Microplate_assay_XylB.png" src="https://static.igem.org/mediawiki/parts/4/4f/Microplate_assay_XylB.png" height="407" width="684"> </td> | ||
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− | <div><b>Figure 3</b> Plot of the NAD<sup>+</sup> assay. <i> | + | <div><b>Figure 3</b> Plot of the NAD<sup>+</sup> assay. <i>xylB</i> shows activity</div> |
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Latest revision as of 03:38, 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)
![https://static.igem.org/mediawiki/parts/f/f1/TU_Darmstadt_EG_XylB-xylC.png](https://static.igem.org/mediawiki/parts/f/f1/TU_Darmstadt_EG_XylB-xylC.png)
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.
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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.
In a NAD+ assay activity of xylB the activity of xylB could been proven
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 significant production in E. coli.
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Figure 2
Scan of the PAGE containing four different samples: marker (M; Protein Marker III AppliChem); samples 1-4 reference and induced. |
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Figure 3 Plot of the NAD+ assay. xylB shows activity
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Figure 4: HPLC-MS spectra from cell lysate with artificialy added ethylene glycol |
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Figure 5: Our culture which was induced with xylose showed no ethyleneglycol after extraction in mass spectrometrie |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
1. Liu H, Ramos KR, Valdehuesa KN, Nisola GM, Lee WK, Chung WJ. Biosynthesis of ethylene glycol in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(8):3409-17.
2. Toivari MH, Nygard Y, Penttila M, Ruohonen L, Wiebe MG. Microbial D-xylonate production. Appl Microbiol Biotechnol. 2012;96(1):1-8.