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Abstract:

One of the main problematics that is always connected with manufacturing is the utilization of the (produced) products after their usage is not needed anymore. This problem affects the industry and the research. Our this year’s project deals with the solution of this issue as we are going to covering  on the one hand the industrial site by producing (biodegradable prostheses) . On the other hand the research site is represented by the general usage of esterified polymers and their efficient decomposition to usable monomers.  In our approach, we the iGEM-Team TU Darmstadt 2015 want to solve this issue by improvement of already existing processes that take place in vitro. This approach combines results out of three years of iGEM. The methods we are going to focus on were developed within the TU Darmstadt projects from 2012, 2014 and 2015.

Xylan degradation

The 2015 project “Building with light” party assesses on the cleavage of the Hemicellulose yylane which is contained in rooted plants as well as in algae. The occurring main repetition unity within this hteropolysaccharide is the aldopentose xylose. The degradation of xylane is accomplished by the usage the enzyme xynA (BBa_K1602038)  for decomposition of xylan main chains as well the enzymes aes (BBa_K1602039) for acetyl group decomposition. To optimize this decomposition step the enzmes are linked to a scaffold construction that was introduced in TU Darmstadt project 2014 (). Thereby the main carbon-source for our host E.coli and thus for the in vivo part is provided.

Monomer production

Adjacent on this step the production of our three monomers itaconic acid, ethylene glycol and xylitol is achieved by implementation of three parts. Thereby the making of itaconic acid and xylitol is done by the enzymes cis-aconitate decaboxlase (BBa_K1602006)  and and heterologous aldose reductase (BBa_K1602005) . In contrast the production of ethylene glycol requires the expression of xylB (BBa_K1602009)  for metabolization of the carbon source xylose.  The overexpression of the already in E.coli occurring gene xylC (BBa_K1602010)  serves the prevention of a possible bottleneck.

Polymer decomposition

Subsequent to the in vivo part as well as the chemistry and P&P part the decomposition of our manufactured prostheses is realized by the Humicola insolens cutinase (HIC) (BBa_K1602020) .  The Cutinase as an α/β-hydrolase  posseses two very interesting properties.  It can connect as well as cleave ester bindings which makes it possible for us to conduct composition as well as degradation of existing substrates . This enzyme was introduced and characterized qualitatively as well as quantitatively in iGEM BioBrick registry by TU Darmstadt iGEM team in 2012.

Safety approach

To finally assure the practicability of our project we focused furthermore on the design of a killswitch function within our organisms. The killswitch is controlled on RNA level based on an improvement of the already existing basic part coding for the cell toxin hokD (BBa_K1602055) . Previously switched to hokD are newly invented riboregulator BioBricks RRlocked (BBa_K1602053)  and RRkey (BBa_K1602052) . Both parts together form the killswitch that induces cell-death in presence of arabinose but allows proliferation of the bacteria in a glucose-rich environment.

 

Conclusion:

By interlocking these improved constructs of 2012 and 2014 with our 2015 project we are able to present a highly efficient and self-contained project that covers a wide range of trendsetting approaches started by degradation of a heteropolymer and ending in the composition of a new polymer.