The steroid-inducible pOp6/LhGR gene expression system is fast, sensitive and does not cause plant growth defects in rice (Oryza sativa)

Inducible systems for transgene expression activated by a chemical inducer or an inducer of non-plant origin are desirable tools for both basic plant research and biotechnology. Although, the technology has been widely exploited in dicotyledonous model plants such as Arabidopsis, it has not been optimised for use with the monocotyledonous model species, namely rice. We have adapted the dexamethasone-inducible pOp6/LhGR system for rice and the results indicated that it is fast, sensitive and tightly regulated, with high levels of induction that remain stable over several generations. Most importantly, we have shown that the system does not cause negative growth defects in vitro or in soil grown plants. Interestingly in the process of testing, we found that another steroid, triamcinolone acetonide, is a more potent inducer in rice than dexamethasone. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

The Steroid-Inducible pOp6/LhGR Gene Expression System is Fast, Sensitive and Does NOT Cause Plant Growth Defects in Rice (Oryza Sativa)

Inducible systems for transgene expression activated by a chemical inducer or an inducer of non-plant origin are desirable tools for both basic plant research and biotechnology. Although, the technology has been widely exploited in model plants, it has not been optimised for use with the major monocotyledonous crop species, namely rice. We have adapted the dexamethasone-inducible pOp6/LhGR system for rice and shown that it is fast, sensitive and tightly regulated, with high levels of induction that remain stable over several generations. Most importantly, we have shown that the system does not cause negative growth defects in vitro or in soil grown plants. Interestingly in the process of testing, we found that another steroid, triamcinolone acetonide, is a more potent inducer in rice than dexamethasone. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps how to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.

Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.

AGES: An auxin-inducible, GAL4-compatible, gene expression system for Drosophila

The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila, spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here we describe the Auxin-inducible Gene Expression System (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila.

The steroid-inducible pOp6/LhGR gene expression system is fast, sensitive and does NOT cause plant growth defects in rice (Oryza sativa)

SummaryInducible systems for transgene expression activated by a chemical or an inducer of non-plant origin are desirable tools for both basic plant research as well as advanced biotechnological utilizations. Although, the technology has been widely exploited in model plant species, optimal solution is missing for the major monocotyledonous crop species – rice. Here, we characterise the inducible properties of the pOp6/LhGR adapted for rice as fast, sensitive, tight, with high levels of induction that remain stable over several generations. Most importantly, the system does not cause negative growth defects in vitro or in soil grown plants. We describe various methods of application and optimization of induction by finding out that another steroid, triamcinolone acetonide, is more potent inducer then dexamethasone in rice. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps how to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.Significance statementThe non-monocot codon-optimized version of the dexamethasone inducible pOp6/LhGR system does not cause severe developmental perturbations in rice plants!

Multidimensional single-cell benchmarking of inducible promoters for precise dynamic control in budding yeast

For quantitative systems biology, simultaneous readout of multiple cellular processes as well as precise, independent control over different genes’ activities are needed. In contrast to readout systems such as fluorescent proteins, control systems such as inducible transcription-factor-promoter systems have not been characterized systematically, impeding reliable modeling and precise system-level probing of biological systems.We built a comprehensive single-copy library of inducible promoters controlling fluorescent protein (yEVenus) expression in budding yeast, including GAL1pr, GALLpr, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr and the blue light-inducible systems EL222-LIP, EL222-GLIP. To track their properties under dynamic perturbations, we performed high-throughput time-lapse microscopy. The analysis of >100 000 cell images was made possible by the recently developed convolutional neural network YeaZ. We report key coarse-grained kinetic parameters, levels of noise, and effects on cellular growth. Our multidimensional benchmarking uncovers unexpected disadvantages of widely used tools, e.g., slow off kinetics of the doxycycline-induced tetOpr system, nomonotonic activity, or high variability of PHO5pr. Our data would guide the choice of acceptable compromises for applications. Evaluating the ARG3 promoter for potential use as a new inducible system, we discovered that it has an interesting OR gate function and that it turns on in the presence of methionine in synthetic complete medium. To demonstrate the ability to finely control genetic circuits, we tuned the time between cell cycle Start and mitotic entry in budding yeast experimentally, exogenously simulating near-wild-type timing.The data presented here ought to facilitate the choices of expression systems for quantitative experiments and applications in systems and synthetic biology and to serve as a reference to benchmark new inducible systems.

Confounding factors from inducible systems for spatiotemporal gene expression regulation

Spatiotemporally regulated targeted gene manipulation is a common way to study the effect of gene variants on phenotypic traits, but the Cre/loxP and Tet-On/Tet-Off systems can affect whole-organism physiology and function due to off-target effects. We highlight some of these adverse effects, including whole-body endocrinology and disturbances in the gut microbiome and in mitochondrial and metabolic function.