Difference between revisions of "Team:Bielefeld-CeBiTec/Results"

Cell-free synthesis of proteins (CFPS) is possible and highly advantageous when it comes to biosafety, speed and versatility, since no living cells are required. We established a highly efficient and robust CFPS reaction. Many different factors were optimized during the course of this project. By providing the protocol we hope to enrich the iGEM community with a new chassis: cell-free extract. The protocol is easy to reproduce. The extract can be applied on simple paper and freeze dried to enable long-term storage and elimination of any living cells. Simple rehydration with water is possible, and results in a still active cell extract. By expression of certain transcription factors prior to sonication of the cells, CFPS can be used as an biosensor, if the correct plasmid DNA is provided. Cell-free protein synthesis therefore offers great potential to carry synthetic biology from the lab to the field.

We established a new assay called Plasmid Repressor Interaction Assay (PRIA) for the repression/derepression model system, the lac operon. It is based on detection of the disruption of the binding of a repressor protein to the cognate DNA upon binding of an analyte in vitro. This works as expected for the repressor protein immobilized on Ni-NTA agarose. We were able to detect the release of DNA from LacI upon addition of IPTG. For the implementation of the assay on paper we immobilized amino- and Cy3-labeled DNA containing the operator site for the corresponding protein successfully on common filter paper. We proved with an electrophoretic mobility shift assay that the repressor proteins for the different biosensors fused with sfGFP we cloned and purified can bind to oligonucleotides with the corresponding operator site. We visualized both the fusion protein and Cy3-labeled DNA simultaneously with a fluorescence scanner.

However, these technical applications for biosensors are useless if there is no analyte to be detected! In order to develop a modular test strip, that can detect several substances at once, we developed, respectively improved sensors to detect several heavy metals (arsenic, chromium, copper, lead, mercury and nickel). All of these sensors were characterized in vivo, some of them were characterized in vitro using Cell Free Protein Synthesis as well. We were able to prove that arsenic and mercury sensors work well in vivo. Our sensors for copper and lead showed tendencies to detect concentrations near the limits provided in WHO guidelines for drinking water contaminations.
To tackle a local problem, we additionally established a new sensor to prevent and assess date rape drug intoxications. We used the repressor BlcR that acts on γ hydroxy butyrate 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 CFPS. Successful in vitro characterization was performed. The operator sequence was followed by a sequence coding for super folder green fluorescent protein (sfGFP), enabling us to detect GHB or heavy metals in liquids by analyzing fluorescence signals. With our biosensor, easy, quick and reliable detection of date rape drugs or heavy metals are now within one's reach.

We designed a functional device for fluorescence detection with your smartphone. It consists of two different filters. One is placed in front of your camera, the other one in front of your flash. In a dark environment, like our special black box, you can take pictures of the paper with the CFPS reactions and will be able to see the fluorescence of the produced sfGFP For analysis we programmed an app, because objective analysis of fluorescence is impossible with the bare eye, since you need to quantify and take into account different factors, like the negative effect of heavy metals on the performance of CFPS. The app quantifies the fluorescence and gives out a list of contaminants in your sample.

In order to optimize our test strips and better understand our biological systems, we modeled cell-free protein synthesis and a repressor-based biosensor. The model accurately reflects the results of several wet lab experiments and was used to investigate the influence of crucial design aspects. Possible applications are the optimization of the output signal and adjustment of the sensitivity of our biosensors to detection limits.

We investigated the biosecurity issue dual use in detail. We provided a detailed report about the applying laws, aspects of the ongoing ethical discussion and the approach within the iGEM competition. We propose a biosecurity risk assessment and the implementation of biosecurity, biosafety and dual use definitions in the iGEM safety page - for iGEM to be a role model in a responsible research community. The detailed report contains points of views of various experts. The deep analysis of the biosecurity aspect significantly influenced our decisions and biosecurity measures throughout the project. The detailed report containing guidelines for future teams is available as PDF.

All the BioBricks we build in the course of this project are listed on our parts page. We have some favourites, that you really should meet! And since we were one of the many teams with an output related to fluorescence, we decided to take part in the Interlab Study as well. Here are our exciting results!