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Continuous Measurement of Biological Noise in <em>Escherichia Coli</em> Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Technion - Israel Institute of Technology

As the fields of biotechnology and synthetic biology expand, cheap and sensitive tools are needed to measure increasingly complicated genetic circuits. In order to bypass some drawbacks of optical fluorescent reporting systems, we have designed and created a co-culture microbial fuel cell (MFC) system for electronic reporting. This system leverages the syntrophic growth of Escheriachia. coli (E. coli) and an electrogenic bacterium Shewanella oneidensis MR-1 (S. oneidensis). The fermentative products of E. coli provide a carbon and electron source for S. oneidensis MR-1, which then reports on such activity electrically at the anode of the MFC. To further test the capability of electrical reporting of complicated synthetic circuits, a novel synthetic biological comparator was designed and tested with both fluorescent and electrical reporting systems. The results suggest that the electrical reporting system is a good alternative to commonly used optical fluorescent reporter systems since it is a non-toxic reporting system with a much wider dynamic range.

A Novel Bioelectronic Reporter System in Living Cells Tested with a Synthetic Biological Comparator.

Bioluminescence is visible light produced and emitted by living cells using various biological systems (e.g. luxCDABE cassette). Today, this phenomenon is widely exploited in biological research, biotechnology and medical applications as a quantitative technique for the detection of biological signals. However, this technique has mostly been used to detect a single input only. In this work, we re-engineered the complex genetic structure of luxCDABE cassette to build a biological unit that can detect multi-inputs, process the cellular information and report the computation results. We first split the luxCDABE operon into several parts to create a genetic circuit that can compute a soft minimum in living cells. Then, we used the new design to implement an AND logic function with better performance as compared to AND logic functions based on protein-protein interactions. Furthermore, by controlling the reverse reaction of the luxCDABE cassette independently from the forward reaction, we built a comparator with a programmable detection threshold. Finally, we applied the redesigned cassette to build an incoherent feedforward loop that reduced the unwanted crosstalk between stress-responsive promoters (recA, katG). This work demonstrates the construction of genetic circuits that combine regulations of gene expression with metabolic pathways, for sensing and computing in living cells.

Synthetic metabolic computation in a bioluminescence-sensing system.

The generation of information, energy and biomass in living cells involves integrated processes that optimally evolve into complex and robust cellular networks. Protein homo-oligomerization, which is correlated with cooperativity in biology, is one means of scaling the complexity of protein networks. It can play critical roles in determining the sensitivity of genetic regulatory circuits and metabolic pathways. Therefore, understanding the roles of oligomerization may lead to new approaches of probing biological functions. Here, we analyzed the frequency of protein oligomerization degree in the cell proteome of nine different organisms, and then, we asked whether there are design trade-offs between protein oligomerization, information precision and energy costs of protein synthesis. Our results indicate that there is an upper limit for the degree of protein oligomerization, possibly because of the trade-off between cellular resource limitations and the information precision involved in biochemical reaction networks. These findings can explain the principles of cellular architecture design and provide a quantitative tool to scale synthetic biological systems.

Ramez Daniel

Department of Biomedical Engineering

Technion - Israel Institute of Technology

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

Ramez Daniel

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

Technion - Israel Institute of Technology

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

Department of Biomedical Engineering