Methods in molecular biology最新文献

Golden Gate cloning allows rapid and reliable assembly of multiple DNA fragments in a defined orientation. Golden Gate cloning requires careful design of the restriction fragment overhangs to minimize undesired products and to generate the desired junctions. The ApE (A plasmid Editor) software package can assist in silico design of input fragments or to generate expected assembly products.

CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. While currently offering the most affordable source of synthetic DNA, oligo pools also come with limitations such as a maximum synthesis length (approximately 350 bases), a higher error rate compared to alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single-guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries.

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria.

Golden Gate cloning enables the modular assembly of DNA parts into desired synthetic genetic constructs. The "one-pot" nature of Golden Gate reactions makes them particularly amenable to high-throughput automation, facilitating the generation of thousands of constructs in a massively parallel manner. One potential bottleneck in this process is the design of these constructs. There are multiple parameters that must be considered during the design of an assembly process, and the final design should also be checked and verified before implementation. Doing this by hand for large numbers of constructs is neither practical nor feasible and increases the likelihood of introducing potentially costly errors. In this chapter we describe a design workflow that utilizes bespoke computational tools to automate the key phases of the construct design process and perform sequence editing in batches.

Synthetic biology, also known as engineering biology, is an interdisciplinary field that applies engineering principles to biological systems. One way to engineer biological systems is by modifying their DNA. A common workflow involves creating new DNA parts through synthesis and then using them in combination with other parts through assembly. Assembly standards such as MoClo, Phytobricks, and Loop are based on Golden Gate, and provide a framework for combining parts. The Synthetic Biology Open Language (SBOL) has implemented a best practice for representing build plans to communicate them to other practitioners through whiteboard designs and in a machine-readable format for communication with lab automation tools. Here we present a software tool for creating SBOL representations of build plans to simulate type IIS-mediated assembly reactions and store relevant metadata.

Bio-Layer Interferometry (BLI) is a technique that uses optical biosensing to analyze interactions between molecules. The analysis of molecular interactions is measured in real-time and does not require fluorescent tags. BLI uses disposable biosensors that come in a variety of formats to bind different ligands including biotin, hexahistidine, GST, and the Fc portion of antibodies. Unlike surface plasmon resonance (SPR), BLI is an open system that does not require microfluidics, which eliminates issues that result from clogging and changes in viscosity. Importantly, BLI readings can be completed in minutes and can be formatted for high throughput screening. Here we use biotinylated short chain phosphoinositides and phosphatidic acid bound to streptavidin BLI biosensors to measure the binding of the soluble Qc SNARE Vam7 from Saccharomyces cerevisiae. Unlike most SNAREs, Vam7 lacks a transmembrane domain or lipid anchor to associate with membranes. Instead Vam7 associates to yeast vacuolar membranes using its N-terminal PX domain that binds to phosphatidylinositol 3-phosphate (PI3P) and phosphatidic acid (PA). Using full length Vam7, Vam7^Y42A, and PX domain alone, we determined and compared the dissociation constants (K_D) of each to biotinylated PI3P and PA biosensors.

Metabolomics data analysis includes, next to the preprocessing, several additional repetitive tasks that can however be heavily dataset dependent or experiment setup specific due to the vast heterogeneity in instrumentation, protocols, or also compounds/samples that are being measured. To address this, various toolboxes and software packages in Python or R have been and are being developed providing researchers and analysts with bioinformatic/chemoinformatic tools to create their own workflows tailored toward their specific needs. This chapter presents tools and example workflows for common tasks focusing on the functionality provided by R packages developed as part of the RforMassSpectrometry initiative. These tasks include, among others, examples to work with chemical formulae, handle and process mass spectrometry data, or calculate similarities between fragment spectra.

Hox genes are highly conserved developmental regulators instrumental to the formation of a wide range of diverse body plans across metazoans. While significant progress in the field of Hox gene research has been made, persistent challenges in unraveling their mechanisms of action and full repertoire of functions remain. To date, investigations of Hox gene function have been primarily conducted in research models belonging to ecdysozoa and vertebrata. Herein we summarize recent findings on Hox genes' roles in the asexual reproduction of the regenerative flatworm planaria, a member of the understudied superphylum Spiralia. We detail our optimized methods for planarian culture, gene perturbation, and induction of asexual reproduction. We aim to provide an experimentally tractable means to dissect Hox gene adult tissue functions underlying planarian asexual reproduction with broader relevance to Hox genes' established and emerging roles in regulating cellular behaviors, developmental patterning, animal behavior, and tissue regeneration.