isopropyl-thiogalactoside and 4-epianhydrotetracycline

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches.

Topics: Arginine; Argininosuccinate Lyase; Bacterial Proteins; Base Pairing; Base Sequence; Citrulline; Corynebacterium glutamicum; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Gene Expression Regulation, Bacterial; Gene Targeting; Glucose-6-Phosphate Isomerase; Isopropyl Thiogalactoside; Plasmids; RNA, Guide, Kinetoplastida; Tetracyclines

Synthetic biology is an emerging branch of molecular biology that uses synthetic genetic constructs to create man-made cells or organisms that are capable of performing novel and/or useful applications. Using a synthetic chemically sensitive genetic toggle switch to activate appropriate fluorescent protein indicators (GFP, RFP) and a cell division inhibitor (minC), we have created a novel E. coli strain that can be used as a highly specific, yet simple and inexpensive chemical recording device. This biological "nanorecorder" can be used to determine both the type and the time at which a brief chemical exposure event has occurred. In particular, we show that the short-term exposure (15-30 min) of cells harboring this synthetic genetic circuit to small molecule signals (anhydrotetracycline or IPTG) triggered long-term and uniform cell elongation, with cell length being directly proportional to the time elapsed following a brief chemical exposure. This work demonstrates that facile modification of an existing genetic toggle switch can be exploited to generate a robust, biologically-based "nanorecorder" that could potentially be adapted to detect, respond and record a wide range of chemical stimuli that may vary over time and space.

Topics: Escherichia coli; Genes, Reporter; Genetic Engineering; Green Fluorescent Proteins; Isopropyl Thiogalactoside; Luminescent Proteins; Nanostructures; Plasmids; Red Fluorescent Protein; Synthetic Biology; Tetracyclines; Time Factors

Accurately predicting noise propagation in gene networks is crucial for understanding signal fidelity in natural networks and designing noise-tolerant gene circuits. To quantify how noise propagates through gene networks, we measured expression correlations between genes in single cells. We found that noise in a gene was determined by its intrinsic fluctuations, transmitted noise from upstream genes, and global noise affecting all genes. A model was developed that explains the complex behavior exhibited by the correlations and reveals the dominant noise sources. The model successfully predicts the correlations as the network is systematically perturbed. This approach provides a step toward understanding and manipulating noise propagation in more complex gene networks.

Topics: Bacterial Proteins; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Fluorescence; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Reporter; Green Fluorescent Proteins; Isopropyl Thiogalactoside; Lac Repressors; Luminescent Proteins; Mathematics; Microscopy, Fluorescence; Models, Genetic; Promoter Regions, Genetic; Red Fluorescent Protein; Repressor Proteins; Signal Transduction; Stochastic Processes; Tetracyclines; Transcription, Genetic; Viral Proteins; Viral Regulatory and Accessory Proteins

A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expression is much more tightly regulated than for the conventional multicopy expression vectors. The simplest version of these copy-control expression vectors is based on the pBAC/oriV plasmid that carries the trfA up-mutant gene under control of the l-arabinose-inducible Para promoter (araC-PBAD). The same promoter controls expression of a gene cloned into MCS. Thus, addition of the inducer (l-arabinose) simultaneously turns on amplification of the plasmid and expression of the cloned gene. Net result is about a 50,000-fold increase in the cloned gene expression. However, when not induced, background expression level is very low, which is important for the maintenance of any "toxic" genes. This vector could be used in most E. coli hosts. Similar versions of the described vector employ the rhamnose-inducible Prha promoter (rhaS-Prha). Other expression systems allow independent regulation of the plasmid amplification and of the cloned gene expression, and some also use the PLtetO-1 promoter. Copy-control expression vector pETcoco, based on the pT7lacO promoter, is commercially available.

Topics: Chromosomes, Artificial, Bacterial; Cloning, Molecular; Gene Dosage; Genes, Reporter; Genetic Vectors; Isopropyl Thiogalactoside; Plasmids; Promoter Regions, Genetic; Rhamnose; Tetracyclines

Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.

Topics: Arabinose; Base Sequence; Cloning, Molecular; DNA-Cytosine Methylases; Escherichia coli; Gene Expression Regulation, Bacterial; Isopropyl Thiogalactoside; Kinetics; Lac Operon; Molecular Sequence Data; Promoter Regions, Genetic; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Restriction Mapping; Tetracycline Resistance; Tetracyclines; Transcription, Genetic