Difference between revisions of "Part:BBa K1602006"
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| + | <h2>cadA Assay</h2> | ||
| + | The decarboxylic activity of cadA was shown via a pH indicator assay. As a byproduct of the catalytic conversion of cis-aconitate to itaconic acid carbon dioxide is released into the assay mixture. Here it forms carbonic acid and thereby lowers the pH value of the mixture. This is visualized through the indicator bromothymol blue (BTB) which changes its configuration state depending on the surrounding pH-Value (pH 6.0 - 7.6). That change of configuration can be shown via a photometric analysis in a TECAN® Infinite 200 PRO microplate reader. The resulting data sheets are then put into a plotting script written in R and exported as a ggplot. | ||
| + | <br> | ||
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| + | The assay was performed as following:<br> | ||
| + | First Na<sub>2</sub>HPO<sub>4</sub> was adjusted to a pH of 7.0 to function as a buffer. The final concentration of Na<sub>2</sub>HPO<sub>4</sub> was 5mM. The assay system contained 10% (v/v) indicator stock (BTB) and 20 ul were added per well. As a negative control a purified TES protein fraction from disrupted BL21 cells was used. In a range from 0mM to 1mM (0,2mM steps; total of 6 reactions) cis-aconitate (substrate), dissolved in Na<sub>2</sub>HPO<sub>4</sub>-buffer solution was added per well to enable the enzymatic conversion. To put the turnover into relation with the maximum turnover we added extra rows that contained equal amounts of substrate and product (itaconic acid). Finally a row containing just itaconic acid was put at the bottom to be able to use the resulting values as a blank for the assay. | ||
| + | <br><br> | ||
| + | The 96 well microplate was loaded as depicted in the picture below: | ||
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| + | <table> | ||
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| + | <td align="center"> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/thumb/0/08/Da15_cadA_plate_assay.png/800px-Da15_cadA_plate_assay.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/thumb/0/08/Da15_cadA_plate_assay.png/320px-Da15_cadA_plate_assay.png"> | ||
| + | </a> | ||
| + | </td> | ||
| + | <td valign="top"> | ||
| + | The assay was run for 200 kinetic cycles, each 30 secs long and with 25 photo pulses per cycle. The reader was heated to the appropriate temperature of 37° celsius. Absorbance was measured at the absorption maximum of BTB which in this case is 620nm. | ||
| + | </td> | ||
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| + | <b>Figure 5</b> 96-well microplate layout | ||
| + | </td> | ||
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| + | </td> | ||
| + | </tr> | ||
| + | </colgroup> | ||
| + | </table> | ||
| + | </div> | ||
</body> | </body> | ||
Revision as of 16:24, 18 September 2015
Inducible itaconic acid producing construct (only cadA)
Itaconic acid is an organic, dicarboxylic acid that is biotechnologically synthesized most commonly in Aspergillus terreus. It is derived from citric acid via 2 intermediates and a final decarboxylation.To enable this pathway in Escherichia coli it is necessary to introduce 1 genes. This gene is taken from the genome Apergillus terreus. It is coding for a cis-aconitate decarboxylase (cadA). An Enzyme that catalyzes the following chemical reaction:
Figure 1 Reaction scheme of the itaconic acid producing operon (only cadA). The substrate for the reaction is cis-aconitate. Cis-aconitate is metabolized to itaconic acid in 1 step by decarboxylation.
Usage
This part is a composite of one coding gene, provided with a strong RBS (BBa_B0034) and under control of a T7 Promoter (BBa_I719005). Optimization of this operon may be possible through introduction of 2 more genes. Namely gltA (BBa_K1602001) and acnA (BBa_K1602002).
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| Figure 2 Genetic map of the itaconic acid producing operon (only cadA) with T7 promoter. This brick enables E.Coli BL21 cells to synthesize itaconic acid in presence of the inductor IPTG. |
Results
The expression of cadA has been visualized via SDS-PAGE. Positive clones were grown at 37° celsius until an OD of 0,7. Afterwards the cells were induced utilizing 20µl of 1M IPTG for 12h at 28° celsius. Finally the cells were lysated via ultrasonic cell disruption.
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Figure 3 Scan of the PAGE containing from left to right a marker (M; Protein Marker III AppliChem), the positive sample (1) and a negative control (2). The picture was cropped and edited for clarification purposes.
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Figure 4 Plot of the gel lanes based on contrast analyses - created with ImageJ
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cadA Assay
The decarboxylic activity of cadA was shown via a pH indicator assay. As a byproduct of the catalytic conversion of cis-aconitate to itaconic acid carbon dioxide is released into the assay mixture. Here it forms carbonic acid and thereby lowers the pH value of the mixture. This is visualized through the indicator bromothymol blue (BTB) which changes its configuration state depending on the surrounding pH-Value (pH 6.0 - 7.6). That change of configuration can be shown via a photometric analysis in a TECAN® Infinite 200 PRO microplate reader. The resulting data sheets are then put into a plotting script written in R and exported as a ggplot.The assay was performed as following:
First Na2HPO4 was adjusted to a pH of 7.0 to function as a buffer. The final concentration of Na2HPO4 was 5mM. The assay system contained 10% (v/v) indicator stock (BTB) and 20 ul were added per well. As a negative control a purified TES protein fraction from disrupted BL21 cells was used. In a range from 0mM to 1mM (0,2mM steps; total of 6 reactions) cis-aconitate (substrate), dissolved in Na2HPO4-buffer solution was added per well to enable the enzymatic conversion. To put the turnover into relation with the maximum turnover we added extra rows that contained equal amounts of substrate and product (itaconic acid). Finally a row containing just itaconic acid was put at the bottom to be able to use the resulting values as a blank for the assay.
The 96 well microplate was loaded as depicted in the picture below:
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The assay was run for 200 kinetic cycles, each 30 secs long and with 25 photo pulses per cycle. The reader was heated to the appropriate temperature of 37° celsius. Absorbance was measured at the absorption maximum of BTB which in this case is 620nm. |
| Figure 5 96-well microplate layout |