Single-cell kinetics of a repressilator when implemented in a single-copy plasmid

Abstract

Synthetic genetic clocks, such as the Elowitz-Leibler repressilator, will be key regulatory components of future synthetic circuits. We constructed a single-copy repressilator (SCR) by implementing the original repressilator circuit on a single-copy F-plasmid. After verifying its functionality, we studied its behaviour as a function of temperature and compared it with that of the original low-copy-number repressilator (LCR). Namely, we compared the period of oscillations, functionality (the fraction of cells exhibiting oscillations) and robustness to internal fluctuations (the fraction of expected oscillations that would occur). We found that, under optimal temperature conditions, the dynamics of the two systems differs significantly, although qualitatively they respond similarly to temperature changes. Exception to this is in the functionality, in which the SCR is higher at lower temperatures but lower at higher temperatures. Next, by adding IPTG to the medium at low and high concentrations during microscopy sessions, we showed that the functionality of the SCR is more robust to external perturbations, which indicates that the oscillatory behaviour of the LCR can be disrupted by affecting only a few of the copies in a cell. We conclude that the SCR, the first functional, synthetic, single-copy, ring-type genetic clock, is more robust to lower temperatures and to external perturbations than the original LCR. The SCR will be of use in future synthetic circuits, since it complements the array of tasks that the LCR can perform.

Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures

“…Either of these possibilities is physically possible since lowering temperatures could affect the efficiency of repressors (see e.g. 48 ), DNA packaging (known to differ between plasmid and chromosomes 49 , or DNA super-coiling 35 (known to affect both packaging 10 and transcription 19,30,50,51 ).…”

Hardware, Software, and Wetware Codesign Environment for Synthetic Biology

“…However, de novo synthesis of large sequences, e.g., the yeast chromosome, is costly, timeconsuming, complex to engineer, and problematic due to spurious depurination of synthesized oligomers [86,87] that causes sequence polymorphisms. To cope with costs, time, and sequence defects, researchers have attempted to design multiple DNA devices and transform them into live cells using molecular biology tools employed in an automated [88] instead of manual [89] method. In particular, automated approaches use standardized, scalable, modular cloning techniques for fast, high-throughput construction of multiple DNA devices and systems simultaneously from shared libraries of DNA parts [90].…”

“…To cope with costs, time, and sequence defects, researchers have attempted to design multiple DNA devices and transform them into live cells using molecular biology tools employed in an automated [ 88 ] instead of manual [ 89 ] method. In particular, automated approaches use standardized, scalable, modular cloning techniques for fast, high-throughput construction of multiple DNA devices and systems simultaneously from shared libraries of DNA parts [ 90 ].…”

“…Image and data analysis workflows have been developed to measure and track the fluorescence intensity of single cells or an entire cell population for multiple cell generations over time. As required, peripheral, inverted microscopes, equipped with white brightfield or fluorescence brightfield, are used for, potentially, multichannel, multilocation, time-lapse imaging generation [ 89 , 108 ]. In these experiments, external variables and experimental conditions can test and observe cells trapped in various external contexts.…”