Difference between revisions of "Team:TU Darmstadt/Demonstrate"

Proof of Concept

Glycolic acid production in E. coli

Our aim was to produce glycolic acid for the production of biodegradable polymers. We were able to overexpress the genes aceA and ycdW in Escherichia coli. The proteins AceA and YcdW were his-tagged and purified via an ÄKTA system. The purification was confirmed by an SDS-PAGE and a western blot. After that, we successfully characterized YcdW with an NADPH dependent enzyme assay. The assay indicates that YcdW uses glyoxylate as substrate under consumption of NADPH to produce glycolic acid. The functionality of AceA was verified with a phenylhydrazine-dependent assay. AceA catalyzes the reaction from isocitrate to glyoxylate. The enzyme assays were performed a second time over a longer incubation period and analyzed via HPLC. The expected product of the reaction was found, indicating a catalytic activity of the purified enzymes. When the enzyme YcdW was produced in E.Coli, the formation of glycolic acid was observed in the disrupted cells (Figure 1). The combined evidence shows, that the transformation of E.Coli was successful, leading to the production of functional enzymes. These enzymes then partake in the production pathway of glycolic acid, which was shown in vivo, as well as in vitro.

Figure 1: HPLC analysis of supernatant of disrupted ycdW transformed E. coli cells. Glyoxylate and glycolic acid peaks can be seen at 12.066 minutes and 15.568 minutes.

Glycolic acid production in S. cerivisiae

As glycolic acid is one monomer which is needed to produce PLGA our goal was to produce glycolic acid in S. cerevisiae. It was possible for us to successfully express AtGLYR1 in S. cerevisiae. This was shown by western blot analysis as well as during an enzyme assay. Using a NADPH dependent enzyme assay we could proof the functionality of our purified enzyme. This is shown in Figure X. During the assay AtGLYR1 converts glyoxylate to glycolic acid while turning over NADPH. Therefore, we were able to produce glycolic acid in vitro. Furthermore, we were able to delete several genes from the genome of CEN.PK 1C. This could be helpful to increase the production of glycolic acid in vivo.

Manufacturing of polymer

noch zu korrigieren

The fact, that we were able to manufacture polymers like PLGA and PLGC is proved by the GPC analysis. The GPC of PLGC (I) shows a slim distribution of a molecule weight of about 12,000 g/mol, which is, without a doubt, a evidence for polymer.

Und hierhin den Graphen der GPC