Difference between revisions of "Part:BBa K1602017"
(18 intermediate revisions by 2 users not shown) | |||
Line 11: | Line 11: | ||
<h1><small>D</small>-xylonic acid producing operon</h1> | <h1><small>D</small>-xylonic acid producing operon</h1> | ||
− | <small>D</small>-Xylose is a monosaccharide belonging to the aldopentose family. It was recently shown that the <small>D</small>-xylose dehydrogenase <i>xylB</i> from <i>Caulobacter crescentus</i> can convert <small>D</small>-xylose to <small>D</small>-xylonolactone. This can react spontaneously or through the catalysation of <i>xylC</i> to <small>D</small>-xylonic acid. | + | <small>D</small>-Xylose is a monosaccharide belonging to the aldopentose family. It was recently shown that the <small>D</small>-xylose dehydrogenase <i>xylB</i> from <i>Caulobacter crescentus</i> can convert <small>D</small>-xylose to <small>D</small>-xylonolactone. This can react spontaneously or through the catalysation of <i>xylC</i> to <small>D</small>-xylonic acid. (2) |
<br> | <br> | ||
<br> | <br> | ||
− | In <i>E. coli</i> <small>D</small>-xylonic acid can further be metabolized to ethyleneglycol by the enzymes yjhG <a href="https://parts.igem.org/Part:BBa_K1602012">(BBa_K1602012)</a> yagE <a href="https://parts.igem.org/Part:BBa_K1602011">(BBa_K1602011)</a> and yqhD <a href="https://parts.igem.org/Part:BBa_K1602013">(BBa_K1602013)</a> which are already present in this host.(1) | + | In <i>E. coli</i> <small>D</small>-xylonic acid can further be metabolized to ethyleneglycol by the enzymes yjhG <a href="https://parts.igem.org/Part:BBa_K1602012">(BBa_K1602012)</a>, yagE <a href="https://parts.igem.org/Part:BBa_K1602011">(BBa_K1602011)</a> and yqhD <a href="https://parts.igem.org/Part:BBa_K1602013">(BBa_K1602013)</a> which are already present in this host. (1) |
<br> | <br> | ||
Line 62: | Line 62: | ||
<td align="center" valign="middle"> | <td align="center" valign="middle"> | ||
− | <img | + | <img class="transparent" alt="https://static.igem.org/mediawiki/parts/1/12/TU_Darmstadt_Plasmidkarte_T7-B0034-xylB-B0034-xylC.png" width="75%" height="75%" src="https://static.igem.org/mediawiki/parts/1/12/TU_Darmstadt_Plasmidkarte_T7-B0034-xylB-B0034-xylC.png"> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</td> | </td> | ||
</tr> | </tr> | ||
Line 75: | Line 72: | ||
</td> | </td> | ||
<td> | <td> | ||
− | + | ||
</td> | </td> | ||
</tr> | </tr> | ||
Line 89: | Line 86: | ||
===<h2>Results</h2>=== | ===<h2>Results</h2>=== | ||
<html> | <html> | ||
− | + | <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> | ||
+ | <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> | ||
+ | <br> | ||
+ | <br> | ||
<table> | <table> | ||
<colgroup width=50%> | <colgroup width=50%> | ||
<tr> | <tr> | ||
<td align= "center" valign="middle"> | <td align= "center" valign="middle"> | ||
− | + | <img class="transparent" alt="https://static.igem.org/mediawiki/parts/8/8e/TU_Darmstadt_EG_xylBC_PAGE.png" src="https://static.igem.org/mediawiki/parts/8/8e/TU_Darmstadt_EG_xylBC_PAGE.png"> | |
+ | <tr> | ||
+ | <td align="left"> | ||
+ | <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> | ||
</td> | </td> | ||
− | + | </tr> | |
− | + | <br> | |
− | + | <br> | |
− | + | <tr> | |
− | + | <td align= "center" valign="middle"> | |
− | + | <img class="shrinkToFit" alt="https://static.igem.org/mediawiki/parts/9/9a/T7-xylB-xylC_Assay.png" src="https://static.igem.org/mediawiki/parts/9/9a/T7-xylB-xylC_Assay.png" height="432" width="740"> | |
− | + | </tr> | |
+ | <br> | ||
+ | <br> | ||
</td> | </td> | ||
+ | |||
+ | |||
+ | </td> | ||
+ | <tr> | ||
+ | <td align= "center" valign="middle"> | ||
+ | <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> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | |||
+ | <tr> | ||
+ | |||
<td align="left"> | <td align="left"> | ||
− | <div><b>Figure | + | <div><b>Figure 3</b> Plot of the NAD<sup>+</sup> assay. <i>xylB</i> shows activity</div> |
<br> | <br> | ||
<br> | <br> | ||
</td> | </td> | ||
</tr> | </tr> | ||
+ | <tr> | ||
+ | |||
+ | <td align= "bottom" valign="left"> | ||
+ | <div><img class="transparent" alt="<img class="shrinkToFit transparent" alt="https://static.igem.org/mediawiki/parts/4/44/TU_Darmstadt_MS_mit_EG.png" src="https://static.igem.org/mediawiki/parts/4/44/TU_Darmstadt_MS_mit_EG.png" height="407" width="651">"></div> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> <b>Figure 4:</b> HPLC-MS spectra from cell lysate with artificialy added ethylene glycol | ||
+ | </td> | ||
+ | </tr> | ||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | <td align= "bottom" valign="left"> | ||
+ | <br> | ||
+ | <div><img class="shrinkToFit transparent" alt="https://static.igem.org/mediawiki/parts/4/40/TU_Darmstadt_MS_ohne_EG.png" src="https://static.igem.org/mediawiki/parts/4/40/TU_Darmstadt_MS_ohne_EG.png" height="407" width="651"></div> | ||
+ | </td> | ||
+ | |||
+ | <tr> | ||
+ | <td> | ||
+ | <br> | ||
+ | <b>Figure 5:</b> Our culture which was induced with xylose showed no ethyleneglycol after extraction in mass spectrometrie</td> | ||
+ | </tr> | ||
+ | </tr> | ||
+ | |||
</colgroup> | </colgroup> | ||
</table> | </table> |
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.
|
![]() |
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.
![]() |
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 NAD+ assay. xylB shows activity
|
![]() |
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 |
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.