Project description:The red flour beetle, Tribolium castaneum, is an important model insect and agricultural pest. However, many standard genetic tools are lacking or underdeveloped in this system. Here, we present a set of new reagents to augment existing Tribolium genetic tools. We demonstrate a new GAL4 driver line that employs the promoter of a ribosomal protein gene to drive expression of a UAS responder in the fat body. We also present a novel dual fluorescent reporter that labels cell membranes and nuclei with different fluorophores for the analysis of cellular morphology. This approach also demonstrates the functionality of the viral T2A peptide for bicistronic gene expression in Tribolium. To facilitate classical genetic analysis, we created lines with visible genetic markers by CRISPR-mediated disruption of the yellow and ebony body color loci with a cassette carrying an attP site, enabling future φC31-mediated integration. Together, the reagents presented here will facilitate more robust genetic analysis in Tribolium and serve as a blueprint for the further development of this powerful model's genetic toolkit.
Project description:Increasing interest in homoacetogenic bacteria for the production of biochemicals and biofuels requisites the development of new genetic tools for these atypical production organisms. An attractive host for the conversion of synthesis gas or electricity into multi-carbon compounds is Clostridium ljungdahlii. So far only limited achievements in modifying this organism towards the production of industrially relevant compounds have been made. Therefore, there is still a strong need for developing new and optimizing existing genetic tools to efficiently access its metabolism. Here, we report on the development of a stable and reproducible transformation protocol that is applicable to C. ljungdahlii and several other clostridial species. Further, we demonstrate the functionality of a temperature-sensitive origin of replication in combination with a fluorescence marker system as important tools for future genetic engineering of this host for microbial bioproduction.
Project description:MotivationA range of membrane protein modeling tools has been developed in the past 5-10 years, yet few of these tools are integrated and make use of existing functionality for soluble proteins. To extend existing methods in the Rosetta biomolecular modeling suite for membrane proteins, we recently implemented RosettaMP, a general framework for membrane protein modeling. While RosettaMP facilitates implementation of new methods, addressing real-world biological problems also requires a set of accessory tools that are used to carry out standard modeling tasks.ResultsHere, we present six modeling tools, including de novo prediction of single trans-membrane helices, making mutations and refining the structure with different amounts of flexibility, transforming a protein into membrane coordinates and optimizing its embedding, computing a Rosetta energy score, and visualizing the protein in the membrane bilayer. We present these methods with complete protocol captures that allow non-expert modelers to carry out the computations.Availability and implementationThe presented tools are part of the Rosetta software suite, available at www.rosettacommons.org .Contactjulia.koehler.leman@gmail.com.Supplementary informationSupplementary data are available at Bioinformatics online.
Project description:RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report expansion of TimeLapse sequencing (TimeLapse-seq) to the guanosine analogue, 6-thioguanosine (s6G), which can be recoded under RNA-friendly nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics.
Project description:BackgroundProtein data over circadian time scale is scarce for clock transcription factors. Further work in this direction is required for refining quantitative clock models. However, gathering highly resolved dynamics of low-abundance transcription factors has been a major challenge in the field. In this work we provide a new tool that could help this major issue. Bioluminescence is an important tool for gathering data on circadian gene expression. It allows data collection over extended time periods for low signal levels, thanks to a large signal-to-noise ratio. However, the main reporter so far used, firefly luciferase (FLUC), presents some disadvantages for reporting total protein levels. For example, the rapid, post-translational inactivation of this luciferase will result in underestimation of protein numbers. A more stable reporter protein could in principle tackle this issue. We noticed that NanoLUC might fill this gap, given its reported brightness and the stability of both enzyme and substrate. However, no data in plant systems on the circadian time scale had been reported.ResultsWe tested NanoLUC activity under different scenarios that will be important for generating highly quantitative data. These include enzyme purification for calibration curves, expression in transient plant systems, stable transgenic plants and in planta time series over circadian time scales. Furthermore, we show that the difference in substrate use between firefly luciferase and NanoLUC allows tracking of two different reporters from the same samples. We show this by exploring the impact of a BOAp:BOA-NanoLUC construct transformed into a Col-0 CCA1p:FLUC background.ConclusionsWe concluded that NanoLUC reporters are compatible with established instrumentation and protocols for firefly luciferase. Overall, our results provide guidelines for researchers gathering dynamic protein data over different time scales and experimental setups.
Project description:Base editing tools for cytosine to thymine (C-T) conversion enable genome manipulation at single base-pair resolution with high efficiency. Available base editors (BEs) for C-T conversion (CBEs) have restricted editing scopes and nonnegligible off-target effects, which limit their applications. Here, by screening diversified lamprey cytidine deaminases, we establish various CBEs with expanded and diversified editing scopes, which could be further refined by various fusing strategies, fusing at either N-terminus or C-terminus of nCas9. Furthermore, off-target analysis reveals that several CBEs display improved fidelity. Our study expands the toolkits for C-T conversion, serves as guidance for appropriate choice and offers a framework for benchmarking future improvement of base editing tools.
Project description:RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report the first use of nucleoside recoding with a guanosine analogue, 6-thioguanosine (s6G). Using TimeLapse sequencing (TimeLapse-seq), s6G can be recoded under RNA-friendly oxidative nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics.
Project description:The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.