Difference between revisions of "Part:BBa K1602006"
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Revision as of 01:00, 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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Figure 5TEXT
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