Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli

Abstract

The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.

Quantification of the gene silencing performances of rationally-designed synthetic small RNAs

“…Although the measured variables can have an sRNA-and target gene-specific behaviour, the reported procedure and results support the future characterization of novel sRNAs, confirm the effectiveness of the design via the used guidelines, and elucidate quantitative performance-related and contextdependent features never investigated before for such synthetic silencers. Synthetic sRNAs will enable to face different problems in synthetic biology, such as the simultaneous silencing of different pathways in metabolic engineering studies , the silencing of essential or non-essential genes for bacterial physiology research studies (Man et al 2011;Sharma et al 2013) and the tuning of synthetic circuits to engineer repression systems with low-fluctuations or noise in the regulation of target proteins (Levine et al 2007), which is an important feature to control cell-to-cell variability Zucca et al 2015).…”

CRISPR interference as low burden logic inverters in synthetic circuits: characterization and tuning

“…The latter was in turn assembled upstream an RFP reporter gene, driving its expression on a cotransformed high copy plasmid (Figure 1, panel A). The choice of the constitutive promoters with different strengths (such that BBa_J23116<BBa_J23100 [26]) allowed to study the effect of increasing guide expression for several levels of dCas9 in the cell, which was tuned by varying the concentration of HSL in the medium.…”

Mechanistic Models of Inducible Synthetic Circuits for Joint Description of DNA Copy Number, Regulatory Protein Level, and Cell Load

“…In many works considering a constant LuxR production, not changing throughout the experiments [22,23,[31][32][33][34], H(L) is modeled via a Hill equation and the circuit output, y, can be written as in Equation 5…”

“…The resulting data were used to compute S cell as the numeric time derivative of fluorescence, divided by absorbance over time. S cell was averaged in the exponential growth phase, typically occurring at absorbance values between 0.05 and 0.18 [32]. Finally, the average S cell values of the X 3 r strains at the desired inductions were normalized by the S cell value of a reference culture (MG1655-Z1 bearing the BBa_J107029 constitutive RFP expression cassette in the pSB4C5 plasmid) as internal control to obtain S cell in standardized relative units.…”