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Protocol
In Vivo DNA Assembly Using the PEDA Method
Simple and efficient DNA assembly methods have been widely used in synthetic biology. Here, we provide the protocol for the recently developed PEDA (phage enzyme-assisted in vivo DNA assembly) method for direc...
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Protocol
A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display
Directed evolution is an efficient strategy for obtaining desired biomolecules. Since the 1990s, the emergence of display techniques has enabled high-throughput screening of functional proteins. However, class...
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Protocol
Photoacoustic Lifetime Imaging of Hypoxia
We present an approach based on photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO2) in tissue. This method utilizes methylene blue, a dye widely used in clinical applica...
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Protocol
Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy
Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved i...
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Protocol
Quantifying ATP-Independent Nucleosome Chaperone Activity with Single-Molecule Methods
The dynamics of histone-DNA interactions govern chromosome organization and regulates the processes of transcription, replication, and repair. Accurate measurements of the energies and the kinetics of DNA bind...
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Protocol
Single-Molecule Imaging of the Phase Separation-Modulated DNA Compaction to Study Transcriptional Repression
Liquid-liquid phase separation (LLPS) often induces the formation of biomolecule condensates at the cellular level. The importance of this phenomenon has been demonstrated in many important biological function...
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Protocol
Modeling Plant Transcription Factor Networks Using ConSReg
Plants have developed complex regulatory programs to respond to various environmental stress such as heat, drought, and cold. Systematic understanding of these biological processes depends on robust constructi...
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Protocol
Identification of Plant Co-regulatory Modules Using CoReg
Regulatory network is often characterized by complex interactions between transcription factors (TFs) and target genes. The synergistic regulations among multiple TFs may co-induce/co-suppress the expressions ...
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Protocol
High-Throughput γ-H2AX Assay Using Imaging Flow Cytometry
The γ-H2AX assay is a sensitive and reliable method to evaluate radiation-induced DNA double-strand breaks. The conventional γ-H2AX assay detects individual nuclear foci manually, but is labor-intensive and ti...
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Protocol
Targeted Activation of Arabidopsis Genes by a Potent CRISPR–Act3.0 System
The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repu...
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Protocol
A Surgical Approach to Hindlimb Suspension: A Mouse Model of Disuse-Induced Atrophy
Hindlimb suspension is a well-established rodent model of disuse-induced atrophy and is commonly used to simulate the effects of bed rest and space flight on humans. Over the decades, this method has undergone...
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Protocol
Bioinformatics Tools for Bulk Gene Expression Deconvolution in Diabetic Retinopathy
Retinal neovascularization is one of the leading causes of vision loss and a hallmark of proliferative diabetic retinopathy (PDR). The immune system is observed to be involved in the pathogenesis of diabetic r...
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Protocol
Methods to Study Antagonistic Activities Among Oral Bacteria
Most bacteria in nature exist in multispecies communities known as biofilms. In the natural habitat where resources (nutrient, space, etc.) are usually limited, individual species must compete or collaborate w...
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Protocol
Delivery of CRISPR-Cas12a Ribonucleoprotein Complex for Genome Editing in an Embryogenic Citrus Cell Line
Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it i...
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Protocol
PAM-Less CRISPR-SpRY Genome Editing in Plants
Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and ...
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Protocol
Base Editing in Poplar Through an Agrobacterium-Mediated Transformation Method
CRISPR-Cas9 systems have revolutionized genome editing in plants and facilitated gene knockout and functional genomic studies in woody plants, like poplar. However, in tree species, previous studies have only ...
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Protocol
Detection and Analysis of Microtubule Nucleator γ-Tubulin Ring Complex
Golgi-derived microtubules constitute an asymmetrical microtubule network that drives polarized transport of vesicles to support cell polarization and directional migration. Golgi-based microtubule nucleation ...
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Protocol
Assessment of Mitochondrial Dysfunctions After Sirtuin Inhibition
Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone ...
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Protocol
Expression, Purification, and Structure Determination of Human PTCH1–HH-N Complexes
Patched-1 (PTCH1), a tumor suppressor, serves as the receptor of Hedgehog (HH) ligand and negatively regulates the HH signaling pathway. Mutations of PTCH1 are implicated in many human cancers. Structural inve...
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Protocol
Electrogenerated Chemiluminescence Method for Determination of 5-Hydroxymethylcytosine in DNA
5-methylcytosine (5-mC) is regarded as the fifth base of DNA, while 5-hydroxymethylcytosine (5-hmC) is regarded as the sixth base of DNA. With the catalysis of ten–eleven translocation proteins, 5-mC can be co...
