Team:TU Darmstadt/Project/Bio/InVitroDegradation/sec3
Contents
Polyester degradation
Abstract
Since our bioreactor concept is suitable for more pathways than xylan degradation, we searched for another approach to show the functionality of our concept.
3D printing leads to additional plastic creation and eventually to more garbage. The photopolymer synthesized in the course of this project numbers among the group of polyesters - polymeric structures containing ester functional groups. Since a few decades some of these compounds had become widely manufactured synthetics. Due to rare biodegradable capacity, pollution with plastic garbage became a serious problem. Reflecting this problem led us to the idea of inverting the intended polyester production by implementing polyester degradation in the designed in vitro bioreactor.
3D printing leads to additional plastic creation and eventually to more garbage. The photopolymer synthesized in the course of this project numbers among the group of polyesters - polymeric structures containing ester functional groups. Since a few decades some of these compounds had become widely manufactured synthetics. Due to rare biodegradable capacity, pollution with plastic garbage became a serious problem. Reflecting this problem led us to the idea of inverting the intended polyester production by implementing polyester degradation in the designed in vitro bioreactor.
Goal
It is not only our aim to create an efficient, photocurable polymer but also in the long run preserve ecologic values and degrade the resulting polymers eco-friendly. Therefore we insisted on improving a part from last year, the HiC - human insulens cutinase. This protein is capable of cleaving ester bonds and thereby capable of degrading common polymers. The HiC serin hydrolase has a catalytic centrum formed as a catalytic triade. In comparison to other
Results
Final volume of the plate was 200µl containig:
- 142µl Buffer (Na2HPO4 pH 7.0)
- bromothymol blue 10% (v/v)
- TES protein fraction HiC (ranging from 5µl to 30µl; concentration unknown)
- 8µl prepolymer (dissolved in Triton100 and DMSO 1:50)
- TES protein fraction (from BL21 cells ranging from 5µl to 30µl; as a control)
The negative control was done by just adding buffer to the well.
The 96 well microplate was loaded as depicted in the picture below: </p>
The assay was run in a in a TECAN® Infinite 200 PRO multi plate reader for 100 kinetic cycles, each 5 mins long and with 25 photo pulses per cycle. The reader was heated to the appropriate temperature of 42° celsius. Absorbance was measured at the absorption maximum of BTB which in this case is 620nm.
Pictures of the multi plate
