Generator
Part:BBa_K1602006
Designed by: Alexandra Goretzki, Tanja Habeck, Nina Kuschik-Maczollek, Laurin Monnheimer, Steven Pilger, Bianca Reisinger, Daniel Stumpf Group: iGEM15_TU_Darmstadt (2015-09-08)
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 a SDS-PAGE:
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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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