| Autor: |
Tschirhart T; Fischell Department of Bioengineering, §Institute for Bioscience and Biotechnology Research, ⊥Department of Mathematics, University of Maryland , College Park, Maryland 20742, United States., Zhou XY; Fischell Department of Bioengineering, §Institute for Bioscience and Biotechnology Research, ⊥Department of Mathematics, University of Maryland , College Park, Maryland 20742, United States., Ueda H, Tsao CY; Fischell Department of Bioengineering, §Institute for Bioscience and Biotechnology Research, ⊥Department of Mathematics, University of Maryland , College Park, Maryland 20742, United States., Kim E, Payne GF; Fischell Department of Bioengineering, §Institute for Bioscience and Biotechnology Research, ⊥Department of Mathematics, University of Maryland , College Park, Maryland 20742, United States., Bentley WE; Fischell Department of Bioengineering, §Institute for Bioscience and Biotechnology Research, ⊥Department of Mathematics, University of Maryland , College Park, Maryland 20742, United States. |
| Abstrakt: |
In order to match our ability to conceive of and construct cells with enhanced function, we must concomitantly develop facile, real-time methods for elucidating performance. With these, new designs can be tested in silico and steps in construction incrementally validated. Electrochemical monitoring offers the above advantages largely because signal transduction stems from direct electron transfer, allowing for potentially quicker and more integrated measurements. One of the most common genetic reporters, β-galactosidase, can be measured both spectrophotometrically (Miller assay) and electrochemically. However, since the relationship between the two is not well understood, the electrochemical methods have not yet garnered the attention of biologists. With the aim of demonstrating the utility of an electrochemical measurement to the synthetic biology community, we created a genetic construct that interprets and reports (with β-galactosidase) on the concentration of the bacterial quorum sensing molecule autoinducer-2. In this work, we provide a correlation between electrochemical measurements and Miller Units. We show that the electrochemical assay works with both lysed and whole cells, allowing for the prediction of one from the other, and for continuous monitoring of cell response. We further present a conceptually simple and generalized mathematical model for cell-based β-galactosidase reporter systems that could aid in building and predicting a variety of synthetic biology constructs. This first-ever in-depth comparison and analysis aims to facilitate the use of electrochemical real-time monitoring in the field of synthetic biology as well as to facilitate the creation of constructs that can more easily communicate information to electronic systems. |
