Team:Bielefeld-CeBiTec/Results
Our Project in a Nutshell
We constructed functional biosensors for various substances that work in a lyophilized in vitro transcription/translation system applied onto paper. Different factors were modeled to enhance the functionality of our cell-free protein synthesis (CFPS) and implemented successfully. Furthermore, we decided to work with a second cell-free approach as well, to further increase speed and durability. For this a functional protocol for this Plasmid Repressor Interaction Assay (PRIA) was established.
The fluorescence output could be analyzed by our newly developed color filter based system with a conventional smartphone and our self-programmed app. CFPS and our output signal processing are nearly universally applicable for BioBrick based biosensors and thus allow for diversification of the biosensors. We constructed biosensors for heavy metals, because many iGEM projects have already focused on them and we aimed at the simultaneous detection of various substances at once. Additionally, we also detect date rape drugs, which are used with increasing frequency, and a quick test of your beverage can be extremely useful.
In 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". The results were implemented in our biosecurity aspects. To further improve our experiments, we interviewed several experts in the course of the project.
Cell-free synthesis of proteins (CFPS) is 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 is produced by sonication of cells and subsequent addition of certain supplements. It 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 and subsequent addition of plasmid DNA with special features, CFPS can be used as a biosensor. Cell-free protein synthesis therefore offers great potential to carry synthetic biology from the lab to the field.
Additionally, we established a new assay called Plasmid Repressor Interaction Assay (PRIA). Basically, the separation of a repressor protein from the corresponding DNA, triggered by the interaction with a specific substance, is detected in vitro. For the implementation of the assay on paper, we immobilized aminolabeled DNA containing the operator site for the corresponding protein successfully on common filter paper. The repressor proteins for the different biosensors fused with sfGFP were proven to bind to the operator sites. PRIA is advantageous over conventional biosensors, because signals can be detected 15 min after addition of the sample and the risk of releasing genetically modified organisms is not existent, since it works only with purified DNA and purified protein.
However, these technical construction forms of biosensors are useless without an analyte to detect! In order to show the versatility of our biosensors we developed a modular test strip for the detection of several substances at once. We newly developed, respectively improved biosensors to detect several heavy metals (arsenic, chromium, copper, lead, mercury and nickel). We characterized all these sensors 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 combined our findings with CFPS by performing successful in vitro characterization.
In each biosensor the operator sequence was followed by a sequence coding for super folder green fluorescent protein (sfGFP), enabling us to detect γ hydroxy butyrate (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.
To further simplify the usage of our biosensors, we designed a functional device for fluorescence detection with your smartphone. Two different color filters are used. One is placed in front of your camera, the other one in front of your flash. In a dark environment, like our special black case, you can take pictures of the fluorescent test strip paper with the CFPS reactions. Since objective analysis of fluorescence is impossible for the bare eye, we provide a self-programmed app for signal analysis. The app takes 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 biosensors 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, like the optimization of the output signal and adjustment of the sensitivity of our biosensors to specific limits.
In order to further investigate the influences of our project on our environment, we investigated the biosecurity issue dual use in detail. This is related to our project, because we were dealing with dangerous substances, that are available for the broad public. We wanted to know, in which way information about these issues could cause harm. We provided a detailed report on dual use covering 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 built in the course of this project are listed on our parts page. 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.
