The optimization system for preparation of TG1 competent cells and electrotransformation

Chai, Dafei; Wang, Gang; Fang, Lin; Li, Huizhong; Liu, Shanshan; Zhu, Haiying; Zheng, Junnian

doi:10.1002/mbo3.1043

Cited by 10 publications

(8 citation statements)

References 14 publications

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“…The previously prepared plant extracts were then evaluated for their potential antibacterial activity against three strains of Gram-negative Escherichia coli species, more exactly, TG1 (a derivative strain of E. coli JM101, which has neither modification nor restriction on transformed exogenous DNA) [ 61 ], DH5-α (a typical engineered E. coli widely used in the laboratory) [ 62 ], and E410A5 (corresponding to E. coli construct used in molecular biology) [ 63 ]. Using Luria-Bertani broth, Miller, the TG1, DH5-α, and E410A5 strains were grown overnight at 37 °C with 160 revolutions per minute [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

Hydrogel for the Controlled Delivery of Bioactive Components from Extracts of Eupatorium glutinosum Lam. Leaves

et al. 2023

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This research reported a hydrogel loaded with the ethanolic and methanolic extracts of Eupatorium glutinosum Lam. The E. glutinosum extracts were characterized by phytochemical screening, Fourier-transform infrared spectroscopy (FTIR), thin-layer chromatography (TLC), and UV/Vis profile identification. This research also evaluated the pharmacological activity of the extracts using antimicrobial, antioxidant, and anti-inflammatory assays prior to polymeric encapsulation. Results indicate that extracts inhibit the Escherichia colii DH5-α (Gram negative) growth; excellent antioxidant activity was evaluated by the ferric reducing power and total antioxidant activity assays, and extracts showed an anti-hemolytic effect. Moreover, the cotton and microcrystalline cellulose hydrogels demonstrate successful encapsulation based on characterization and kinetics studies such as FTIR, extract release, and swelling degree. Moreover, effective antibacterial activity was registered by the loaded hydrogel. The overall results encourage and show that Eupatorium glutinosum-loaded hydrogel may find a wide range of bandage and wound healing applications in the biomedical area.

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Section: Methodsmentioning

confidence: 99%

Hydrogel for the Controlled Delivery of Bioactive Components from Extracts of Eupatorium glutinosum Lam. Leaves

et al. 2023

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“…It is also necessary to resuspend the cells very carefully at each stage, since they are very fragile and easily destroyed. It is recommended to store the obtained cells at -80 °C (Chai et al, 2020). At this temperature, the cells retain their competence for a longer time.…”

Section: Competent Cellsmentioning

confidence: 99%

Problems of creating antibody phage libraries and their solutions

Aripov,

Volkova,

Ilyichev

et al. 2024

Vestn. VOGiS

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Phage display has become an efficient, reliable and popular molecular technique for generating libraries encompassing millions or even billions of clones of divergent peptides or proteins. The method is based on the correspondence between phage genotype and phenotype, which ensures the presentation of recombinant proteins of known amino acid composition on the surface of phage particles. The use of affinity selection allows one to choose variants with affinity for different targets from phage libraries. The implementation of the antibody phage display technique has revolutionized the field of clinical immunology, both for developing tools to diagnose infectious diseases and for producing therapeutic agents. It has also become the basis for efficient and relatively inexpensive methods for studying protein–protein interactions, receptor binding sites, as well as epitope and mimotope identification. The antibody phage display technique involves a number of steps, and the final result depends on their successful implementation. The diversity, whether natural or obtained by combinatorial chemistry, is the basis of any library. The choice of molecular techniques is critical to ensure that this diversity is maintained during the phage library preparation step and during the transformation of E. coli cells. After a helper phage is added to the suspension of transformed E. coli cells, a bacteriophage library is formed, which is a working tool for performing the affinity selection procedure and searching for individual molecules. Despite the apparent simplicity of generating phage antibody libraries, a number of subtleties need to be taken into account. First, there are the features of phage vector preparation. Currently, a large number of phagemid vectors have been developed, and their selection is also of great importance. The key step is preparing competent E. coli cells and the technology of their transformation. The choice of a helper phage and the method used to generate it is also important. This article discusses the key challenges faced by researchers in constructing phage antibody libraries.

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“…[63] were improved for B. animalis by adding 0.5 M sucrose to the MRS growth medium and washing buffer, and adding citrate to the electroporation buffer [64] . Transformation of B. bifidum was only possible after addition of 16% FOS or 10% GOS to the growth media and growing the bacterial cells to late log phase [65] . Electrotransformation efficiencies for E. coli TG1 were improved by increasing the culture time (8-10 hrs), the bacterial cell concentration (OD 600 = 0.45) and the culture volume [66] .…”

Section: Improving Transfer Efficiency: Electroporationmentioning

confidence: 99%

Establishing genetic manipulation for novel strains of human gut bacteria

Sheridan¹,

Odat²,

Scott³

2023

Microbiome Res Rep

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Recent years have seen the development of high-accuracy and high-throughput genetic manipulation techniques, which have greatly improved our understanding of genetically tractable microbes. However, challenges remain in establishing genetic manipulation techniques in novel organisms, owing largely to exogenous DNA defence mechanisms, lack of selectable markers, lack of efficient methods to introduce exogenous DNA and an inability of genetic vectors to replicate in their new host. In this review, we describe some of the techniques that are available for genetic manipulation of novel microorganisms. While many reviews exist that focus on the final step in genetic manipulation, the editing of recipient DNA, we particularly focus on the first step in this process, the transfer of exogenous DNA into a strain of interest. Examples illustrating the use of these techniques are provided for a selection of human gut bacteria in which genetic tractability has been established, such as Bifidobacterium, Bacteroides and Roseburia. Ultimately, this review aims to provide an information source for researchers interested in developing genetic manipulation techniques for novel bacterial strains, particularly those of the human gut microbiota.

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The optimization system for preparation of TG1 competent cells and electrotransformation

Cited by 10 publications

References 14 publications

Hydrogel for the Controlled Delivery of Bioactive Components from Extracts of Eupatorium glutinosum Lam. Leaves

Hydrogel for the Controlled Delivery of Bioactive Components from Extracts of Eupatorium glutinosum Lam. Leaves

Problems of creating antibody phage libraries and their solutions

Establishing genetic manipulation for novel strains of human gut bacteria

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