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Results Overview
Here you can find the results of all experimental aspects of our project, starting with the Troygenic assembly, endocytosis and CeDIS. These parts of the project include the cloning of several plasmids. Thereupon, (the) model, measurement, and hardware, each of which greatly contributed to the respective aspects of the construction of the Troygenics, are presented. Finally, we demonstrate our results and achievements, and present an outlook on further research possibilities.
Pivotal for our project is the construction of our Troygenics. To achieve this, we designed two plasmids and co-transformed them into E. coli which than produce Troygenics. Afterwards, we demonstrated the correct assembly with different methods, including atomic force microscopy, ddPCR and nanopore sequencing.
One of the key issues we had to face was to get our Troygenic selectively internalized into the target cell. We were able to prove specific endocytosis of several cell specific ligands by fusing them to the fluorescence reporter mCherry and observing the uptake via fluorescence measurements.
Our varieties of Cell Death Inducing System (CeDIS) consist of different Cas13a systems in combination with guide RNAs. The CeDIS results in collateral RNA cleavage and leads to cell death. We have demonstrated the functionality of Lbu and Lsh with growth experiments.
Using our model, we identified the optimal split points for split antibiotic resistances by implementing structural- and 3D-modeling. We verified this model in wet lab experiments of a split-chloramphenicol resistance.Furthermore, we calculated the adequate number of gRNAs for lab experiments, using a mathematical modeling approach including growth curve modeling.
To get a deeper insight into the behavior of our model organisms, we designed microfluidic chips, which we produced using 3D-printing. On these chips we cultivated S. cerevisiae and A. niger. We also designed and built a low-cost microfluidic laboratory starter kit to make microfluidics easily accessible for other teams.
To enable the generation of scientifically sound data, it is necessary to standardize all measurements. In the context of life sciences, it is of uttermost importance to achieve this for fluorescence data. After the iGEM measurement committee had established fluorescein as a reference for all GFP measurements, we conferred the same principle to mCherry measurements. We introduced Texas Red as a reference fluorophore for several mCherry applications.
We demonstrated that every part of our Troygenic workes as designed. Firstly, we showed the correct assembly of the Troygenic. Thereafter, we showed receptor induced endocytosis of specific ligands for S. cerevisiae and A. niger. Moreover, we demonstrated that our Cas13a-based CeDIS is working. Finally, we have successfully assembled a Troygenic which was able to transform S. cerevisiae.
On this page, we describe the possible approaches to optimize the effectivity of our Troygenics and the potentials of customizing our system for various applications in the future.