Cell-free synthetic biology
Many questions in cell biology were answered by biochemistry studies on purified molecular machines and enzymes. The advantage of experiments on minimal, defined systems is that their components and their respective concentrations are known, and can be adjusted easily.
In my lab we don’t limit ourselves to reconstituting isolated molecular processes that exist in nature. Our goal is to construct artificial, life-like systems from simple biochemical and synthetic building blocks. Using this approach we aim to gain an improved fundamental understanding of natural biological systems and to develop new materials and reaction systems that may find applications in bio- and nanotechnology.
Unlike classical cell-free reaction systems, cells are not merely “soups” of reactants. They arrange, separate and control reactions in their interior by confining biochemical processes to subcellular locations and compartments. How much spatial organization is necessary to integrate multiple complex biochemical functions in cell-free systems or cell mimics? To answer this question, we are interested in engineering new, programmable materials to provide spatial organization in cell-free reaction systems.
Another question is how living organisms achieve spatial organization. Our understanding of self-organization in biology is still far from complete and not advanced enough to take advantage of its mechanisms in engineering. We use cell-free systems to investigate self-organization and self-assembly processes. The simplified and well-controlled conditions we can achieve in cell-free reactions combined with microfluidic technology are a great advantage.
Oscillating biochemical networks provide the rhythms for many important cellular processes such as the circadian clock and mitosis. Synthetic gene networks can generate sustained oscillations of gene expression in cell-free reactions in microfluidic nanoreactors (Niederholtmeyer et al. 2013, Niederholtmeyer et al. 2015). We use oscillators as model systems to study how to engineer circuits with robust dynamics. In the future, these circuits will also become useful to reliably time biochemical processes in cell-free systems and cell mimics.
As compared to living cells, cell-free systems have a number of properties that simplify experiments, quantitative measurements and models: they are decoupled from growth or viability requirements, open to direct manipulation and less complex. Cell-free transcription and translation (TX-TL) systems achieve the flow of information from DNA to protein in a test tube. TX-TL experiments speed up design-build-test cycles by eliminating time-consuming cloning steps so that proteins and genetic networks can be tested more rapidly. We have previously demonstrated that gene networks engineered in TX-TL can be successfully transferred to E. coli and that in vitro and in vivo results matched closely (Niederholtmeyer et al. 2015). Cell-free experiments have the potential to fill the gap between theoretical models and studies in cells to accelerate the engineering of biology.
We use and develop microfluidic technology for our experiments. Microfluidic devices reduce reagent consumption and enable precision control of fluids and reactions. For example, they can sustain long-term steady state conditions in TX-TL reactions. By emulating the far-from-equilibrium conditions that cells maintain by exchanging energy and matter with the environment, microfluidic platforms, in combination with TX-TL, have even allowed the assembly and characterization of dynamic oscillating gene networks and produce results that closely match in vivo circuit dynamics (Niederholtmeyer et al. 2015). Microfluidic tools also enable the controlled and rapid assembly of uniform biomimetic structures such as the membranes and organelles of cell-mimics (Niederholtmeyer et al. 2018).
Cell-free transcription and translation (TX-TL) systems produce mRNA and protein from a DNA template like a plasmid or simply a PCR product. We use TX-TL systems based on an E. coli lysate or a completely defined TX-TL system consisting of the purified, reconstituted translation machinery of E. coli.
Synthetic and hybrid materials
To assemble new systems with life-like functionalities we are not limited to biological materials. Combinations of natural and synthetic materials will lead to new functions not found in nature as well as improved engineerability and stability.