Prokaryotes

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    Protocols in Current Issue
    Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter
    Authors:  Yuki Yamanaka, Hiroki Watanabe, Erika Yamauchi, Yukari Miyake and Kaneyoshi Yamamoto, date: 01/20/2020, view: 1203, Q&A: 0
    [Abstract] The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced ...
    Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA
    Authors:  Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 2446, Q&A: 0
    [Abstract] Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...
    High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach
    Authors:  Franziska M. Heydenreich, Tamara Miljuš, Dalibor Milić and Dmitry B. Veprintsev, date: 01/05/2020, view: 1732, Q&A: 0
    [Abstract] Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries ...
    Strand-specific Single-stranded DNA Sequencing (4S-seq) of E. coli genomes
    Authors:  Takahiro Masuda, Nobuaki Kono, Masaru Tomita and Kazuharu Arakawa, date: 08/05/2019, view: 2403, Q&A: 0
    [Abstract] Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although ...
    Bacterial Microcolonies in Gel Beads for High-throughput Screening
    Author:  Yolanda Schaerli, date: 07/05/2018, view: 4908, Q&A: 0
    [Abstract] High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.
    ...
    Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
    Authors:  Prarthana Mohanraju, John van der Oost, Martin Jinek and Daan C. Swarts, date: 05/05/2018, view: 8644, Q&A: 3
    [Abstract] This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for ...
    Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases
    Authors:  Zhen Xu, Yan-Ning Rui, John P. Hagan and Dong H. Kim, date: 02/05/2018, view: 5169, Q&A: 0
    [Abstract] Protein tagging is a powerful tool for performing comprehensive analyses of the biological functions of a protein of interest owing to the existence of a wide variety of tags. It becomes indispensable in some cases, such as in tracking protein dynamics in a live cell or adding a peptide epitope due to the lack of optimal antibodies. However, ...
    Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
    Authors:  Enrico König, Francesca Zerbini, Ilaria Zanella, Davide Fraccascia and Guido Grandi, date: 01/20/2018, view: 17714, Q&A: 1
    [Abstract] With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the ...
    Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning
    [Abstract] This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible ...
    Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli
    Authors:  Xin-tian Li, Cindy Sou and Suckjoon Jun, date: 10/05/2017, view: 6361, Q&A: 0
    [Abstract] We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and ...



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