Figure 1. Cell-free systems allow rapid and extensive characterization of biological systems.
Schematic representation of the design-build-test cycle using the cell-free system (top). A design is first modeled to obtain intuition about the architecture. Parts are then assembled on linear DNA without cloning, and tested in vitro. With functional parts, circuit variants can then be tested and working circuits can be extensively characterized. Final circuits are cloned onto plasmids and implemented in vivo. For a specific example of the cell-free system applied to engineering a 5-node oscillator network see Figure 1. Bottom shows a comparison of the time required for testing a genetic circuit by the cell-free approach versus traditional engineering in cells.
DOI: http://dx.doi.org/10.7554/eLife.09771.003
Figure 1—source data 1. Comparison of a Test Cycle in TX-TL vs. a Test Cycle in traditional prototyping.
Shown are time estimates per step for each process. Assumption is that modular PCR fragments exist that can be assembled into Linear DNA. Parts used from Sun et al., (2014) Supplementary Table S2.
elife-09771-fig1-data1.xlsx (39.3KB, xlsx)
DOI: 10.7554/eLife.09771.004
Figure 1—figure supplement 1. Engineering a 5-node negative feedback oscillator using the cell-free framework.
A novel network architecture, which shows the intended behavior in silico is first assembled on linear DNA using in vitro characterized parts. Initial circuit testing on linear DNA is advantageous because: (I) linear DNA can be synthesized in a few hours, (II) it allows rapid testing of multiple circuit variants, (III) and allows expression strengths of network components to be easily tuned by varying their relative concentrations. A functional circuit can then be further characterized to identify parameter ranges that support the desired behavior and to experimentally test hypotheses. If an in vivo implementation is intended, the cloned plasmids are verified for correct function in vitro before in vivo implementation.