An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency

Novel one step transformation method for Escherichia coli and Staphylococcus aureus using arginine-glucose functionalized hydroxyapatite nanoparticles

Competent cultures of Bacillus subtilis 168 were fractionated on gradients of Renografin-76 to obtain a population enriched for competent cells. The cells in this fraction contained two nuclear bodies. The competent cell fraction synthesized deoxyribonucleic acid and ribonucleic acid at reduced rates compared to the noncompetent cell fraction and appeared to divide synchronously upon incubation. The state of the chromosome in competent cells was determined by density transfer experiments and marker frequency analyses. The results are consistent with a competent cell possessing two, or a multiple of two, chromosomes, one complete and the other partially duplicated. During subsequent growth the partially completed chromosome replicates preferentially.

Phage SP50 excludes phage SPP1 both in infection and in transfection of B. subtilis. The dependence of the efficiency of exclusion on the concentration of SP50 DNA shows that one SP50 DNA molecule within a competent cell is sufficient to exclude SPP1 phage development. The concentration dependence allows a determination of the efficiency of uptake of SP50 DNA by competent cells. Only 1 out of 200 SP50 DNA molecules in the transfection mixture will become biologically active in excluding SPP1 phage development in the competent cell.

According to the change of environment, soil-dwelling Bacillus species differentiate into distinct subpopulations, such as spores and competent cells. Rap-Phr systems have been found to be involved in this differentiation circuit by interacting with major regulatory proteins, such as Spo0A, ComA, and DegU. In this study, we report that the plasmid-born RapQ-PhrQ system found in Bacillus amyloliquefaciens B3 affects three regulatory pathways in the heterologous host Bacillus subtilis. Expression of rapQ in B. subtilis OKB105 strongly suppressed its sporulation efficiency, transformation efficiency, and surfactin production. Co-expression of phrQ or addition of synthesized PhrQ pentapeptide in vitro could compensate for the suppressive effects caused by rapQ. We also found that expression of rapQ decreased the transcriptional level of the sporulation-related gene spoIIE and surfactin synthesis-related gene srfA; meanwhile, the transcriptional levels of these genes could be rescued by co-expression of phrQ and in vitro addition of PhrQ pentapeptide. Electrophoretic mobility shift (EMSA) result also showed that RapQ could bind to ComA without interacting with ComA binding to DNA, and PhrQ pentapeptide antagonized RapQ activity in vitro. These results indicate that this new plasmid-born RapQ-PhrQ system controls sporulation, competent cell formation, and surfactin production in B. subtilis OKB105.

Yeast-based interaction assays to determine protein-protein and protein-nucleic acid interactions commonly rely on the reconstitution of chimeric transcription factors that activate the expression of target reporter genes. The enzyme β-galactosidase (β-gal), coded by the LacZ gene of Escherichia coli, is a widely used reporter in yeast systems, and its expression is commonly assessed by evaluating its activity. X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) is an inexpensive and sensitive substrate of β-gal, whose hydrolysis results in an intensely blue colored and easily detectable end product, 5,5'-dibromo-4,4'-dichloro-indigo. The insoluble nature of this end product, however, makes X-gal-based assays unsuitable for direct spectrophotometric absorbance quantification. As such, the use of X-gal is mostly restricted to solid-support approaches, such as colony lift or agar plate assays, which often only provide a qualitative readout. In this article, we describe a quantitative solid-phase X-gal assay to measure protein-protein interaction strength in yeast cells using a simple and low-cost experimental setup. We have optimized multiple aspects of the assay, namely sample preparation, reaction time, and quantification method, for speed and consistency. By integrating the use of a freely available ImageJ-based plugin, we have further standardized the assay for reliability and reproducibility. This improved quantitative X-gal assay can be performed in a standard molecular biology lab without the need for any specialized equipment other than an inexpensive and widely accessible smartphone camera. To exemplify the protocol, we provide detailed step-by-step instructions to perform a quantitative X-gal assay to assess the interaction between two Arabidopsis thaliana proteins, SUPPRESSOR OF PHYA-105 1 (SPA1) and PRODUCTION OF ANTHOCYANIN PIGMENT 2 (PAP2). To demonstrate the sensitivity of our assay in detecting weaker interactions, we also compare the results with a liquid-phase assay that uses ONPG (ortho-nitrophenyl-β-galactopyranoside) as a substrate for β-gal. The quantitative X-gal assay described here can easily be adapted for high-throughout interaction studies and protein domain mapping, even in yeast strains with low levels of LacZ expression. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent yeast cells and transformation Alternate Protocol 1: In-house preparation of yeast competent cells for use in lithium acetate (LiAc)-mediated yeast transformation Support Protocol: Long-term storage and revival of frozen yeast strain stocks Basic Protocol 2: Measuring β-galactosidase activity via the quantitative X-gal assay Alternate Protocol 2: Quantification of interaction strength using liquid ONPG assay.

Yarrowia lipolytica is an industrial host organism with incredible potential for metabolic engineering. However, the genetic tools and capacities in this host lag behind those of conventional counterparts. In this study, we sought to increase the transformation efficiency of Y. lipolytica by creating a simple protocol using electroporation. Efficiency was increased by optimizing wash buffers, pre-culture growth time, OD600 of competent cells, voltage, competent cell volume, DNA concentration, and recovery time. The outcome of these optimizations led to a simple protocol with maximum linear fragment transformation efficiency of 1.6×104 transformants per μg DNA and 2.8×104 transformants per μg DNA for episomal plasmid transformation. The protocol presented here is superior to other Y. lipolytica transformation protocols as it requires no lengthy pretreatment and no required carrier DNA to achieve efficiencies on par with, or exceeding, previously reported methods.

Molecular validation of genetically transformed Catharanthus roseus plants via different strains of Agrobacterium tumefaciens

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.

Chicken embryos kept in culture medium were bombarded using a high helium gas pressure biolistic device. To optimize the factors that affect transformation efficiency, the lacZ gene under control of the human cytomegalovirus immediate early enhancer/promoter was used as a reporter gene. There was an inverse relationship between survival rate and transformation efficiency. The best conditions obtained for high embryo survival and high transformation efficiency were achieved with 800 psi helium gas pressure, 500 mmHg vacuum, gold particles, an 8 cm DNA-coated microparticle flying distance to the embryo and embryo placement 0.5 cm from the center of the particle dispersion cone. Under these conditions, transformation efficiency was 100%, survival rate 25% and the number of expression units in the embryo body cells ranged from 100 to 1,000. Expression of green fluorescent protein was also detected in embryos bombarded under optimal conditions. Based on the results obtained, the biolistic process can be considered an efficient method for the transformation of chicken embryos and therefore can be used as a model system to study transient gene expression and tissue-specific promoters.

Sugarcane growth is negatively affected by abiotic stresses. Genetic engineering of sugarcane with abiotic stress-responsive genes has been the method of choice to confer abiotic stress tolerance. In the present work, we optimized callus induction and transformation of the sugarcane variety CP-77-400 with the promoter region of the OsC3H52 gene to analyze its regulatory function under drought and salt stress. Calli were induced using different callus induction media (CIM). The promoter region of the OsC3H52 gene that belongs to the zinc finger protein family was cloned into two different expression vectors i.e. pBI221 and pGreenII0129, which were subsequently transformed to sugarcane calli through agrobacterium and biolistic transformation methods. Among various callus induction media, CIM3 (5 mg L-1 2,4-D + 10% coconut water) showed high callus induction (93%). Biolistic transformation using recombinant pGreenII0129 at 8.5µg/µl concentration resulted in higher efficiency of transformation (28%). While the agrobacterium mediated transformation using recombinant pBI221 plasmid gave 13% transformation efficiency. Amongst the various concentrations of acetosyringone used in agrobacterium mediated transformation, 100 µM showed higher efficiencies (13%) of transformation. Selection of the transformed calli was performed at 25 mg L-1 Hygromycin and 25 mg L-1 Hygromycin + 300 mg L-1 cefotaxime for 15 days, both for biolistic and agrobacterium mediated transformation, respectively. Transformed calli were then successfully confirmed through PCR and GUS expression. The regulatory role of the OsC3H52 promoter in transgenic calli was further evaluated under drought and salt stress. Quantitative real-time PCR analysis of the GUS reporter gene showed high expression in transgenic calli under drought and salt stress conditions. Key words: Abiotic stress, Callus induction, Gene Transformation, GUS Analysis, Saccharum officinarum

Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20-80 fold more than in anther but similar to that of non-embryogenic callus, which was 20-50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity.

Indian jujube (Ziziphus mauritiana Lamarck) is one of the most popular fruits in South China. On 15 August 2024, a soft rot disease affecting Indian jujube fruits, with an incidence of 15%, was found in the market of Qujing City, Yunnan Province, China. The initial symptom was a localized brown rot, which extended to half of the fruits after three days, and eventually all the fruits turned brown and rotted, accompanied by the odor of alcoholic fermentation, with white mycelium overflowing from the rotting parts. The margins of the infected fruits were cut into 0.5×0.5 mm pieces, soaked in 75% ethanol for three minutes, then rinsed three times with sterile water, and placed on potato dextrose agar (PDA) in the dark, 28°C. After 12 hours, thin white mycelium grew around the tissues, and after 24 hours the diameter of the colony reached 7 cm. After 48 hours, the mycelium had grown to fill the 9 cm medium, and after 72 hours, the upper mycelium turning black, and the entire colony becoming grey-black. After transferring the mycelium to new PDA medium for 72 hours, grey-black colonies were formed again after the above process. Morphologically, the sporangiophores measured 1.04 to 1.89 mm in length and 8.88 to 33.12 μm in diameter, with rhizoids and stolons present at their bases. Sporangia were almost spherical, 122 μm (±64 μm, n=50) in diameter. Sporangiospores were round to ovoid, measuring 8.36 to 20.01 × 5.83 to 17.12 μm (mean 9.35 μm, n=50). The morphological characteristics of this isolate were analogous to Rhizopus stolonifer (Haque et al. 2023). To identify this pathogen molecularly, primers ITS1/ITS4 were used to amplify the ITS1-5.8S-ITS2 rDNA region (Zarrin et al. 2016), PCR products were purified and ligated to the vector (pClone007 Versatile Simple Vector), followed by introduction into competent cells (Trelief ® 5α Chemically Competent Cell), coated on LB plates and single clones were picked. Finally, the plasmids were sequenced with universal primers M13F-47/M13R-48 (Tsingke Biotech Co., Ltd., Kunming, China) (James et al. 2006; Woo et al. 2010). The sequence obtained was deposited with accession number PQ557265.1 and was 99.54% homologous with Rhizopus stolonifer var. stolonifer (OP863060.1) in the Genbank database. Five wounded and five non-wounded Indian jujube fruits were inoculated with mycelium grown for 2 days, Another set of five wounded and five non-wounded fruits were inoculated with PDA medium as a control, these were incubated in the dark at 28°C. The wounded fruits inoculated with R. stolonifer exhibited the same morbidity symptoms observed previously, and the entire fruits had become completely rotten after 96 hours. Non-wounded fruits inoculated with R. stolonifer, as well as the control, remained free of disease after 5 days. It is worth noting that when a non-wounded fruit is adjacent to an infected one, the mycelium invades the non-wounded fruit from where the pedicel has fallen off, leading to its infection. The pathogen was reisolated from symptomatic fruits and was identified as R. stolonifer, which fulfilled Koch's postulates. To our knowledge, this is the first report of R. stolonifer causing fruit rot of Indian jujube.

Bacillus subtilis has been widely used as a biological control agent in agricultural production. Environmental strains of B. subtilis are an important source of biological control agents. However, due to its low genetic transformation efficiency, the genetic manipulation of the environmental and nondomesticated strains is challenging. In this study, the impact of competent cell preparation, pulse electroporation, and recovery culture on the electroporation efficiency of B. subtilis GLB191 was assessed utilizing response surface methodology. Results indicated that the concentration of glycine, DL-threonine, and Tween 80 used in a cell wall weakening solution during competent cell preparation, and the voltage applied during pulse electroporation were the primary factors affecting electroporation efficiency. Optimization of these factors led to nearly a three-fold increase (reaching 74.00 ± 5.10 CFU/µg DNA) in electroporation efficiency. The elimination of dam and dcm modifications to mitigate the influence of host restriction-modification systems was integrated to further increase the electroporation efficacy. An electroporation efficiency for replicative plasmids of 1.96 ± 0.05 × 106 CFU/µg DNA was achieved using the optimized strategy. Utilizing this improved methodology, the temperature-sensitive plasmid pJOE8899 was efficiently transformed into B. subtilis GLB191, resulting in a markerless knockout of pdeH. The optimized transformation strategy significantly enhances the efficiency of markerless genome editing of nondomesticated B. subtilis, offering the potential for future interpretation of their modes of action, which is critical for the development of the nondomesticated B. subtilis strains.

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Euphorbia tirucalli evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Optimized parameters resulted in high transformation efficiency 3 fold increase at transient GUS expression .The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (100 µM) and cocultivation temperature of 25°C. Methods: Nodal explants were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56, 0.8 and 1.2 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of NAA 2 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400, 600 and 800 µM. Histochemical analysis of gus gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 72% when the infection process was carried out with acetosyringone in co-cultivation medium (100 µM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

High efficiency transformation of Escherichia coli with plasmids