转化 -微生物遗传学 -微生物学-BIO-PROTOCOL

A New Tool for the Flexible Genetic Manipulation of Geobacillus kaustophilus

RA Ryotaro Amatsu KM Kotaro Mori SI Shu Ishikawa WM Wilfried J. J. Meijer KY Ken-ichi Yoshida*

0 Q&A 787 Views Sep 5, 2022

Geobacillus kaustophilus, a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of Bacillus subtilis can be transferred via pLS20-mediated conjugation resulting in subsequent integration in the genome of G. kaustophilus. Therefore, we have developed a transformation strategy to design an artificial DNA segment on the chromosome of B. subtilis and introduce it into G. kaustophilus. The artificial DNA segment can be freely designed by taking advantage of the plasticity of the B. subtilis genome and combined with the simplicity of pLS20 conjugation transfer. This transformation strategy would adapt to various Gram-positive bacteria other than G. kaustophilus.

Graphical abstract:

Bacterial Conjugation Protocol for Ruminant Mycoplasmas

反刍动物支原体细菌接合的实验方法

ES Eveline Sagné CC Christine Citti ED Emilie Dordet-Frisoni*

0 Q&A 2205 Views Jan 20, 2021

In Mycoplasma agalactiae, two simultaneous processes of DNA transfer have been described that require direct cell-to-cell contact and are similar to conjugation. One involves the self-transmission of an integrative conjugative element (ICE) while the second concerns the horizontal transfer of large and small fragments of chromosomal DNA. Here, we describe an optimized conjugation protocol for the horizontal transfer of ICE or chromosomal DNA carrying antibiotic resistance markers (i.e., tetracycline, gentamicin, puromycin) from donor to recipient mycoplasma cells. Calculation of the conjugation frequencies, selection and characterization of transconjugants are detailed. This protocol has been developed with M. agalactiae but has been successfully used for M. bovis and can be adapted to other related mycoplasma species.

Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka

农杆菌介导的优良籼稻品种Komboka的高效转化

VL Van T. Luu* MS Melissa Stiebner PM Paula Emmerich Maldonado SV Sandra Valdés DM Didier Marín GD Gerardo Delgado VL Virginia Laluz LW Lin-Bo Wu PC Paul Chavarriaga JT Joe Tohme IS Inez H. Slamet-Loedin WF Wolf B. Frommer

0 Q&A 7818 Views Sep 5, 2020

Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world’s population. As a result, numerous transformation methods have been developed for both indica and japonica rice. Since breeders continuously develop new rice varieties, transformation protocols have to be adapted for each new variety. Here we provide an optimized transformation protocol with detailed tips and tricks for a new African variety Komboka using immature embryos. In Komboka, we obtained an apparent transformation rate of up to 48% for GUS/GFP reporter gene constructs using this optimized protocol. This protocol is also applicable for use with other elite indica rice varieties.

Isolation and Characterization of Live Yeast Cells from Ancient Clay Vessels

从古代陶皿中分离鉴定活酵母细胞

Tzemach Aouizerat AM Aren M. Maeir

Yitzhak Paz YG Yuval Gadot AS Amir Szitenberg SA Sivan Alkalay-Oren SC Shunit Coppenhagen-Glazer MK Michael Klutstein*

Ronen Hazan*

0 Q&A 4929 Views Jan 5, 2020

Ancient fermented food has been studied mainly based on residue analysis and recipes and reconstruction attempts were performed using modern domesticated yeast. Furthermore, microorganisms which participated in fermentation were studied using ancient-DNA techniques. In a recent paper, we presented a novel approach based on the hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. Here we present a pipeline for isolation of yeast from clay vessels uncovered in archeological sites and transferred to the microbiology lab where they can be isolated and characterized. This method opens new avenues for experimental archeology and enables attempts to recreate ancient food and beverages using the original microorganisms.

Development of an Efficient Transformation System for Halotolerant Yeast Debaryomyces hansenii CBS767

耐盐汉斯德巴氏酵母菌CBS767的一种高效转化体系的建立

AM Anu P. Minhas* DB Dipanwita Biswas

0 Q&A 3051 Views Sep 5, 2019

Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. Further, its association with traditional cheese and meat products imparting special flavors to these products project this yeast with enormous biotechnological potential in the agrofood sector. However, lack of an efficient transformation system in D. hansenii still direct the complementation based assay in S. cerevisiae mutants for functional analysis of D. hansenii genes. Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain, DBH9 (generated by UV induced random mutagenesis), and the DhHIS4 gene as the selectable marker (Minhas et al., 2009). Moreover, the same method has also been employed for gene disruption in D. hansenii by homologous recombination.

Measurement of the Length of the Integrated Donor DNA during Bacillus subtilis Natural Chromosomal Transformation

枯草芽孢杆菌自然遗传转化过程中完整供体DNA长度的测定

ES Ester Serrano BC Begoña Carrasco*

0 Q&A 3130 Views Aug 20, 2019

For natural transformation to occur, bacterial cells must first develop a programmed physiological state called competence. Competence in Bacillus subtilis, which occurs only in a fraction of cells, is a transient stress response that allows cells to take up DNA from the environment. During natural chromosomal transformation, the internalized linear single-stranded (ss) DNA recombines with the identical (homologous) or partially identical (homeologous) sequence of the resident duplex. The length of the integrated DNA, which can be measured, depends on the percentage of sequence divergence between the donor (internalized) and the recipient (chromosomal) DNAs.

The following protocol describes how to induce the development of competence in B. subtilis cells, how to transform them with donor DNAs representing different percentages of sequence divergence compared with the recipient chromosomal DNA, how to calculate the chromosomal transformation efficiency for each of them, and how to amplify the chromosomal DNA from the transformants in order to measure the length in base pairs (bp) of the integrated donor DNA.

Quantitative Transformation Efficiency Assay for Bacillus subtilis

枯草芽孢杆菌转化效率的定量测定

CL Christian L. Loyo BB Briana M. Burton*

0 Q&A 7443 Views Dec 5, 2018

Bacillus subtilis (B. subtilis) is a model Gram-positive organism used to study a variety of physiological states and stress responses, one of which is the development of competence. Competence refers to the physiological state of a cell which allows it to be transformed naturally. Through induction of competence, the efficiency of natural transformation can be quantified by plating colony forming units (CFU) and transforming units (TFU). Here we describe a protocol for quantifying relative transformability using B. subtilis.

Induction of Natural Competence in Genetically-modified Lactococcus lactis

转基因乳酸乳球菌自然感受态的诱导

Joyce Mulder MW Michiel Wels OK Oscar P. Kuipers MK Michiel Kleerebezem* PB Peter A. Bron

0 Q&A 6471 Views Jul 5, 2018

Natural competence can be activated in Lactoccocus lactis subsp lactis and cremoris upon overexpression of ComX, a master regulator of bacterial competence. Herein, we demonstrate a method to activate bacterial competence by regulating the expression of the comX gene by using a nisin-inducible promoter in an L. lactis strain harboring either a chromosomal or plasmid-encoded copy of nisRK. Addition of moderate concentrations of the inducer nisin resulted in concomitant moderate levels of ComX, which led to an optimal transformation rate (1.0 x 10^-6 transformants/total cell number/g plasmid DNA). Here, a detailed description of the optimized protocol for competence induction is presented.

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

接合实验检测葡萄球菌CRISPR-Cas的抗质粒免疫功能

FW Forrest C. Walker

Asma Hatoum-Aslan*

0 Q&A 11563 Views May 5, 2017

CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. Testing the ability of CRISPR-Cas systems to block plasmid transfer in various conditions or with CRISPR-Cas mutants provides key insights into the functionality and mechanisms of CRISPR-Cas as well as how antibiotic resistance spreads within bacterial communities. Here, we describe a method for quantifying the impact of CRISPR-Cas on the efficiency of plasmid transfer by conjugation. While this method is presented in Staphylococcus species, it could be more broadly used for any conjugative prokaryote.

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

聚乙二醇介导的圆锥掘氏梅里霉的转化

LH Le D. He JE Jonathan J. Ewbank*

0 Q&A 8473 Views Mar 5, 2017

Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used to study host-pathogen interactions. Here, we report a polyethylene glycol-mediated transformation method (Turgeon et al., 2010; Ochman et al., 1988) for this fungus. The protocol can be used to generate both knock-in or knock-out strains (Lebrigand et al., 2016).

微生物学

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