![]() Similar to the backbone 2 versions, the successful cloning was proven by the comparison of the E.coli plates and the restriction digest of the Mini-Preps.įig.4: SDS-Page of the main strain spider silk proteinįurther SDS-PAGE gels will be shown at the Jamboree. The third created backbone 3 version entails four different expression cassettes (Fig. 1 & 2), only differing from each other in the used promoters to create a direct comparison of the efficiency of these promoters. Three different versions of the backbone 3 were obtained, two containing one expression cassette each (Fig. Since the sequence of the insert was already confirmed by sequencing the backbone 1, only the comparison of the E.coli plates and the restriction digest of the Mini-Preps were needed as a evidence for a successful cloning. These samples were again sent to the company “Microsynth” for sequencing.įour backbone 2 versions were created by Golden Gate cloning of the backbone 1 plasmid, proven to obtain the correct sequence. Since no colonies could be observed on the negative control a successful cloning appeared to be likely.Ī further sign for the correct plasmid was given by the control digest, showing bands at the correct sizes for most of the Mini-Prep samples. A first indication for a successful cloning is given by the comparison of the incubated E.coli plates of the negative control with the sample. ![]() The sequences of these PCR-products were also confirmed by sending them for sequencing to the company “Microsynth”.Īfter receiving the correct sequences for the three PCR-products, the DNA-parts were cloned into an empty backbone 1 by Golden Gate. Three further PCR-products, were created out of the first PCR-product, together creating the complete main strain spider silk sequence. After a PCR-product in the desired size was retrieved, it was sent to the company “Microsynth” for sequencing. Since the insert can not be synthesized due to a high number of repeating sequences, it was built together by PCR. We had varying success with out cloning, and will show our progress in the following order: spider silk, PHB and gelatin. Throughout our work, we adjusted our experiments based on troubleshooting according the engineering cycle and after gaining insights during our human practices analyses. Our project encompassed two main approaches: the cloning of coding sequences for our chosen biopolymers into plasmids for Pichia pastoris, and, simultaneously, making a variety of brick mixtures of different sizes and components, and testing them.
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