Team:Bielefeld-CeBiTec/Results
We constructed functional biosensors for date rape drugs, copper and mercury that work in a lyophilized in vitro transcription/translation system applied to paper. Different factors were modelled to enhance the functionality of our cell-free protein synthesis and implemented successfully. We decided to work with a second cell-free approach as well, to further increase speed and durability. A functional protocol for this Plasmid Repressor Interaction Assay was established.
The fluorescence output could be analyzed by our newly developed filter system with a smartphone app we programmed.
Along the course of our project we came across certain pieces of information, which could certainly cause damage if misused. Thus, we decided to put a great effort into the human practices aspect of our project by creating an extensive report on the topic "dual use".
For detailed information on our results, please click into the corresponding areas of our overview picture (noch in Arbeit). For a first short summary on the different topics stay on this page.
Did you know: You do not need living cells to build functional proteins! Cell-free synthesis of proteins is possible and highly advantageous when it comes to biosafety, speed and versatility. We established a highly efficient and robust cell-free synthesis system that is easy to perform. The reaction can be applied on simple paper. Freeze-drying enables long-term storage and elimination of any living cells. Cell-free protein synthesis (CFPS) therefore offers great potential to carry synthetic biology from the lab to the field.
We were able to establish a new assay called the plasmid repressor interaction assay (PRIA) for the model system LacI-lacO. For the implementation of the assay on paper we immobilized amino- and Cy3-labeled DNA containing the operator site successfully on filter paper. We were able to prove with an electrophoretic mobility shift assay that the repressor proteins (ArsR, LacI and BlcR) fused with sfGFP we used can bind to oligonucleotides with the corresponding operator site (arsO, lacO, Pblc). For the detection of the fusion protein and Cy3-labeled DNA we were able to exhibit fluorescence of them both with the Ettan DIGE scanner.
We worked with different sensors to detect several heavy metals (arsenic, chromium, copper, lead, mercury and nickel) which were characterized in vivo. Some of these sensors (arsenic, chromium, copper and mercury) were characterized in vitro using CFPS as well. We were able to prove, that arsenic and mercury sensors work well in vivo while copper and lead showed capability to become sensitive enough to detect WHO guidelines for drinking water contaminations. Especially in combination with our cell free CFPS system we see great potential to improve these.
To prevent and assess date rape drug intoxications, we used the protein BlcR that acts on GHB, a date rape drug ingredient. We proved interaction of this protein from the soil bacterium A. tumefaciens with a specific operator sequence. We combined our findings with a cell-free approach. When the operator sequence was followed by a sequence coding for a reporter protein, we detected GHB in liquids by analyzing fluorescence signals. With our biosensor, easy, quick and reliable detection of date rape drugs and intoxications thereof are now within one's reach.
We designed a functional device for fluorescence detection with your smartphone. You only need additional two filters and a dark environment (or our special black box.) For analysis we programmed an app, because objective analysis is impossible with the bare eye, since you need to take into account different factors, like the effect of heavy metals on the CFPS.
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