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In order to study the effect of energy saving and emission reduction in exterior wall renovation with different thermal insulation materials for residential buildings, taking the existing residential buildings in Jinan as the research object, the model was established by DeST-H energy consumption simulation software to study the influence of four high-performance thermal insulation materials on building energy consumption at different thickeners: expanded polystyrene board (XPS), graphite polystyrene board (SEPS), rigid foam polyurethane (PUR) and rock wool board (RW). The full life cycle method was used to analyze its economy. At the same time, the net present value method was further used to determine the optimal thickness of four kinds of high-performance thermal insulation materials, which were 55, 61, 88 and 38 mm respectively. Finally, the economic benefits and emission reduction effects of the optimal insulation thickness were analyzed. The results show that when the optimal insulation thickness is adopted, the CO2 emission is reduced by 59 759. 93 ~ 61 968. 44 kg, the SO2 emission is reduced by 1 787. 383 ~ 1 864. 65 kg, the NOx emission is reduced by 893. 69 ~ 932. 32 kg, the pollutant emission is reduced by 30% ~ 33%, the environmental benefits ranges from 20 353. 48 yuan to 21 187. 71 yuan. [ABSTRACT FROM AUTHOR]

Sugarcane is a prominent source of sugar and ethanol production. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, long breeding cycle, and recalcitrance to genetic transformation. The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis; however, quick and effective transformation systems are still lacking for sugarcane. Here, we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S. spontaneum. The yield of viable protoplasts was approximately 1.26 × 107 per gram of leaf material, and the transformation efficiency of 80.19% could be achieved under the optimized condition. Furthermore, using this approach, the nuclear localization of an ABI5-like bZIPs transcription factor was validated, and the promoter activity of several putative DNase I hypersensitive sites (DHSs) was assessed. The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays. A highly efficient S. spontaneum mesophyll cell protoplast isolation and transient transformation method was developed, and it shall be suitable for in vivo functional gene analysis in sugarcane.

Bifidobacterium can be used as probiotic that confers various health benefits to its host. The functional analysis of specific genes can help elucidate the probiotic mechanism of Bifidobacterium. However, the low transformation efficiency has hindered the in-depth study of its function by genetic manipulation. In this study, we optimized transformation methods for Bifidobacterium animalis AR668 to improve the transformation efficiency up to 2.15 ×105 CFU/μg DNA. Additionally, the expression of competence gene ssb2 could enhance the transformability which was the first attempt to apply it to B. animalis. Moreover, the transformation efficiency was increased after the plasmids were modified by gene0033 and gene1509 which limited the interference of the restriction-modification system in the uptake of foreign DNA by cells. Based on the above optimization, the transformation efficiency of 7.2 ×105 CFU/μg DNA for AR668 was obtained, which was more than 100 times higher than that of the original condition. Together, we successfully improved the transformation efficiency of B. animalis AR668, contributing to its genetic manipulation and modification. [ABSTRACT FROM AUTHOR]

Antibiotic resistance genes can be spread via gene horizontal transfer (GHT). Chlorination and UV irradiation are common disinfection methods used in wastewater treatment plants before the discharge of treated wastewater. This study aimed to elucidate the effects of disinfection on the transformation of naked DNA in the aquatic environment. The pUC19 plasmid possessing ampicillin-resistant beta-lactamase and subjected to different dosages of chlorine or UV irradiation was used for transformation in Escherichia coli to estimate the transformation efficiency and GHT in the environment after disinfection. The results showed that doses > 0.5 mg-Cl2/L can effectively decrease transformation efficiency (1.21 to 8.83-log10) based on pUC19 as the positive control. UV irradiation can decrease the efficiency (2.37 to 3.39-log10) following 10–60 min of treatment. PCR and qPCR detection have limitations for determining transformation efficiency because they provide approximate estimates damaged DNAs. Overall, these results indicate that proper disinfection management using chlorine and/or UV for treated wastewater before discharge from wastewater treatment plants can prevent the spread of antibiotic resistant bacteria and genes, by decreasing the efficiency of naturally occurring bacterial transformations in wastewater treatment plants.

Detached organs or differentiated tissues could form a mass of pluripotent cells termed as callus on an auxin-rich medium, the underlying molecular mechanism of which remains elusive in cotton. LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor is a key regulator of plant cell totipotency/pluripotency, and a number of cotton GhLBDs with high-level differential expression during the callus induction process have been identified. Their overexpression in cotton calli fostered promotions in and callus induction without exogenous auxin. Expression analysis and histological observation using paraffin sectioning suggested that the first 72 h on culture is a key time point for callus initiation, whereby the GhLBDs showed high transcript abundance and enlarged calli that were rapidly developed from procambium and cambium. GhLBDs' expression level could be precisely modulated by the gradient concentrations of exogenous auxin, whereas auxin transport inhibitor 2,3,5-triiodobenzoic acid could severely inhibit its expression. The LBD-mediated callus formation was also dependent on the expression levels of GhLBDs. Further, a β-estradiol-inducible promoter pER8 was used to drive GhLBD18-1 expression, which led to rapid callus proliferation, suggesting that pER8 / GhLBD18-1 could be used as a selectable marker system to replace the existing antibiotic/herbicide-resistance selectable markers in plant transformation. Our study provides new insights for callus initiation regulatory mechanism and strategies for improving transformation efficiency in cotton. [ABSTRACT FROM AUTHOR]

The antiphage ability is an important feature of fermentation strains in the dairy industry. Our previous work described the bacteriophage exclusion (BREX) system in the probiotic strain, Lactobacillus casei Zhang. The function of L. casei Zhang pglX gene in mediating 5′-ACRCm6AG-3′ methylation was also confirmed. This study aimed to further dissect the function of the BREX system of L. casei Zhang by inactivating its second methyltransferase gene (LCAZH_2054). The methylome of the mutant, L. casei Zhang Δ 2054 , was profiled by single-molecule real-time sequencing. Then, the cell morphology, growth, plasmid transformation efficiency, and stability of the wildtype and mutant were compared. The mutant did not have an observable effect in microscopic and colony morphology, but it reached a higher cell density after entering the exponential phase without obvious increase in the cell viability. The mutant had fewer 5′-ACRCm6AG-3′ methylation compared with the wildtype (1835 versus 1906). Interestingly, no significant difference was observed in the transformation efficiency between the 2 strains when plasmids without cognate recognition sequence (pSec:Leiss:Nuc and pG+host9) were transformed, contrasting to transforming cells with cognate recognition sequence-containing plasmids (pMSP3535 and pTRKH2). The efficiency of transforming pMSP3535 into the LCAZH_2054 mutant was significantly lower than the wildtype, whereas an opposite trend was seen in pTRKH2 transformation. Moreover, compared with the wildtype, the mutant strain had higher capacity in retaining pMSP3535 and lower capacity in retaining pTRKH2, suggesting an unequal tolerance level to different foreign DNA. In conclusion, LCAZH_2054 was not directly responsible for 5′-ACRCm6AG-3′ methylation in L. casei Zhang, but it might help regulate the function and specificity of the BREX system. [ABSTRACT FROM AUTHOR]

The current study was based on Agrobacterium-mediated peanut genetic transformation and effective remediation of transgenic plants. The effect of Agrobacterium tumefaciens strain LBA4404 with a binary vector of PxCSG m-YFP 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene has been evaluated on two commercial varieties and on two sources of explants (Embryo and cotyledon), each containing a CaMV 35S promoter. The comparative regeneration and transformation efficiency analyses revealed that the embryo is a better target tissue than cotyledon, and early-ripe peanut genotypes with relatively small seeds (such as Bard-479) have proven to be comparatively responsive to transformation. Explant culture in media containing 3 mg L 1 indole-3-butyric acid and 0.1 mg L 1 napthalene acetic acid resulted in strong roots of virtually all transgenic plants. More than 87% of the transplanted plants were able to produce morphologically normal blooms and pods containing viable seeds. The phenotypic and genotypic monitoring of the EPSPS gene inheritance over two generations exhibited the desired 3:1 inheritance. Our findings suggest that Agrobacterium-mediated transformation is a feasible and valuable tool for peanut breeding and functional genomics research. [ABSTRACT FROM AUTHOR]

Abstract Background Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism. Results We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA. Conclusions Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.

Abstract Background Passion fruit (Passiflora edulis Sims) is an important horticultural crop in the tropics and subtropics, where it has great commercial potential due to high demand for fresh edible fruits and processed juice as well as source of raw materials in cosmetic industries. Genetic engineering shows great potential in passion fruit improvement and can compensate for the limitations of conventional breeding. Despite the success achieved in genetic modification of few passion fruit varieties, transgenic passion fruit production is still difficult for farmer-preferred cultivars. Therefore, it is important to establish a simple and fast Agrobacterium-mediated cell transformation of commercial hybrid passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa). Results In the present study, we have developed a simple and fast Agrobacterium-mediated transformation system for hybrid passion fruit KPF4 using leaf disc explants. Factors affecting the rate of transient beta (β)-glucuronidase (gusA) expression and consequently transformation efficiency were optimized as follows: Agrobacterium cell density with an OD600 of 0.5, 30 min infection time, 3 days of co-cultivation duration and the incorporation of 200 µM acetosyringone into Agrobacterium infection suspension medium. Using the optimized conditions, transgenic plants of KPF4 were produced within 2 months with an average transformation efficiency of 0.67%. The β-glucuronidase (GUS) histochemical staining confirmed the expression and integration of an intron-containing gusA gene into transformed leaf discs and transgenic plant lines of KPF4. The presence of gusA gene in the transgenic plants was confirmed by polymerase chain reaction (PCR). The results confirmed that the gusA gene was efficiently integrated into the passion fruit genome. Conclusions The developed transformation protocol is simple and rapid and could be useful for functional genomic studies and transferring agronomically important traits into passion fruit hybrid KPF4. This study developed a method that can be used to transfer traits such as resistance to viral diseases, low fruit quality and short storage life. To the best of our knowledge, this is the first report on genetic transformation system for commercial passion fruit hybrid KPF4.

Detached organs or differentiated tissues could form a mass of pluripotent cells termed as callus on an auxin-rich medium, the underlying molecular mechanism of which remains elusive in cotton. LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor is a key regulator of plant cell totipotency/pluripotency, and a number of cotton GhLBDs with high-level differential expression during the callus induction process have been identified. Their overexpression in cotton calli fostered promotions in and callus induction without exogenous auxin. Expression analysis and histological observation using paraffin sectioning suggested that the first 72 h on culture is a key time point for callus initiation, whereby the GhLBDs showed high transcript abundance and enlarged calli that were rapidly developed from procambium and cambium. GhLBDs’ expression level could be precisely modulated by the gradient concentrations of exogenous auxin, whereas auxin transport inhibitor 2,3,5-triiodobenzoic acid could severely inhibit its expression. The LBD-mediated callus formation was also dependent on the expression levels of GhLBDs. Further, a β-estradiol-inducible promoter pER8 was used to drive GhLBD18-1 expression, which led to rapid callus proliferation, suggesting that pER8/GhLBD18-1 could be used as a selectable marker system to replace the existing antibiotic/herbicide-resistance selectable markers in plant transformation. Our study provides new insights for callus initiation regulatory mechanism and strategies for improving transformation efficiency in cotton.

BACKGROUND: Streptococcus thermophilus, one of the most important lactic acid bacteria, is widely used in food fermentation, which is beneficial to improve food quality. However, the current genetic transformation systems are inefficient for S. thermophilus S‐3, which hinders its further study. RESULTS: We developed three electroporation transformation methods for S. thermophilus S‐3, and optimized various parameters to enhance the transformation efficiency up to 1.3 × 106 CFU/μg DNA, which was 32‐fold higher than that of unoptimized. Additionally, transcriptional analysis showed that a series of competence genes in S. thermophilus S‐3 were remarkedly up‐regulated after optimization, indicating that improvement of transformation efficiency was attributed to the expression level of competence genes. Furthermore, to prove their potential, expression of competence genes (comEA, cbpD and comX) were employed to increase transformation efficiency. The maximum transformation efficiency was obtained by overexpression of comEA, which was 14‐fold higher than that of control. CONCLUSION: This is the first report of competence gene expression for enhancing transformability in S. thermophilus, which exerts a positive effect on the development of desirable characteristics strains. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Genetically transformed Bifidobacterium longum , a probiotic bacterium in the human intestines, has a high potential for in-situ delivery of therapeutic proteins. However, one limitation is its low transformation efficiency caused by its thick membrane as it is a Gram-positive bacterium. Here we present an excellent transformation efficiency of B. longum (up to 1 × 107 to 2.1 × 107 cells/μg of DNA), obtained when oxidized multi-walled carbon nanotubes are used in synergy with classic transformation methods. This approach opens a fruitful avenue for B. longum transformations that could increase its potential for in-situ delivery systems of recombinant proteins. [Display omitted] [ABSTRACT FROM AUTHOR]

Paclitaxel (Taxol®) is a potent anticancer agent, but the widespread pharmaceutical use of paclitaxel is hampered by its limited availability due to low accumulation levels in the native yew (Taxus spp.) plants. Currently, hairy root culture is an emerging biotechnological tool that presents several advantages such as reduced costs and higher specialized metabolite production, therefore, its application to paclitaxel production can be of commercial and medicinal interest. The objective of present study was to induce hairy root in Taxus baccata L. by transformation with the wild type Rhizobium rhizogenes A4 strain. Thus, T. baccata was inoculated by three different inoculation methods: (a) ex vitro seedlings inoculation by direct injection of a liquid bacterial culture; (b) ex vitro needles inoculation by liquid co-culturing with bacteria; (c) ex vitro shoots inoculation by dipping liquid bacterial culture. Hairy roots were formed only from ex vitro seedlings inoculated by the direct inoculation method, with transformation efficiency of 14.3%. Formation of hairy roots was observed two months after inoculation. This project forms the basis for the establishment of hairy root cultures from T. baccata for the production of paclitaxel.

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One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of Methylomicrobium buryatense 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered M. buryatense 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous fadE gene in the genome with a heterologous kivd gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs. [ABSTRACT FROM AUTHOR]

One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of Methylomicrobium buryatense 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered M. buryatense 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous fadE gene in the genome with a heterologous kivd gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs.

Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12–16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts.

Catalases are a large group of enzymes that decompose hydrogen peroxide to oxygen and hydrogen, and have been applied widely in numerous areas. Bacillus subtilis ATCC 6051a is a well-known host strain for high level secretion of heterologous peptides. However, the application of 6051a was seriously hampered by insufficient transformation efficiency. In this study, D-xylose inducible comKwas integrated into the genome of B. subtilis ATCC 6051a, generating 164S, amutant owns a transformation efficiency of 1 000-fold higher than its parent strain, thus allowing gene replacement by double crossover recombination using linear dsDNAs. The efficiency of the flanking arms for homologous recombination was then analyzed. We found that 400 bp was the minimal length of homologous fragments required to initiate efficient recombination in the 164S strain. In addition, DNA cassettes encoding two mesophilic catalases (Orf 2-62 and Orf 2-63) from B. licheniformis were integrated onto 164S. The catalytic properties of recombinant Orf 2-62 and Orf 2-63 were analyzed, and were found to be predominantly secreted into the fermentation broth, although they obviously lack any known secretory signal peptide. This work demonstrated that B. subtilis 164S is an excellent cell tool, not only for its superior secretion capacity, but also for its convenience in genetic modification. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

So far, a large number of transformation systems have been established for japonica rice, but only a few have been reported for cold-region varieties. In our study, we established highly efficient tissue culture systems for two cold-region rice cultivars, Dongnong 427 and Longdao 14. Plant growth regulator (PGR) levels were optimized by an orthogonal experimental design. The culture ability, constituted by induction and differentiation rate, served as the detection index of orthogonal experiments. The optimal combinations of PGRs for callus induction and regeneration of Dongnong 427 and Longdao 14 were 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) + 2 mg/l 6-benzyladenine (BA) + 4 mg/l kinetin (KIN) + 0.2 mg/l α-naphthaleneacetic acid (NAA) and 1 mg/l 2,4-D + 4 mg/l 6-BA + 4 mg/l KIN + 0.5 mg/l NAA, respectively. Agrobacterium strain EHA 105 containing the plasmid pCAMBIA1301 was used for transformation. The frequency of transient transformation was expressed as the ratio between the number of calli showing GUS expression and the total number of calli kept for staining. The highest transformation efficiency in Dongnong 427 was obtained when calli were immersed in 0.272 OD600 (optical density determined at 600 nm) for 10 min. While it was best for Longdao 14 calli to be infected with 0.592 OD600 for 20 min. Infected calli of the two varieties were co-cultivated on two pieces of sterile filter paper moistened with 1 ml liquid co-cultivation medium for three days. The expression of the GUS gene was confirmed by PCR analysis of plants of both varieties.

Purslane (Portulaca oleracea L.), a valuable medicinal herb, is used as a source of pharmaceutical components such as flavonoids, alkaloids, fatty acids, terpenoids, and sterols. Regeneration of transgenic purslane plantlets from transformed cells is a time-consuming procedure and needs hard work. In this study, in-planta transformation of purslane, using sonication and vacuum infiltration, is reported. The purslane seeds were infected through Agrobacterium tumefaciens strain LBA4404 having the uidA gene on pBI121 vector. Effective selection of transformants was performed by supplementing MS media with 250 mg l−1 kanamycin. Several factors affecting the in-planta procedure including pre-culture duration, acetosyringone dose, sonication, and vacuum infiltration duration were investigated. The results demonstrated that the highest number of GUS-positive purslane plantlets was obtained when the pre-cultured seeds were sonicated and vacuum-infiltered for 2 min in agro-bacterial cell suspension, and then co-cultivated in MS media having 100 µM acetosyringone. The integration and expression of uidA gene in transgenic purslane was successfully corroborated by southern blot and GUS histochemical analyses. [ABSTRACT FROM AUTHOR]

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

One of the commonly employed methods in molecular biology is to utilize calcium chloride to treat Escherichia coli for the preparation of competent cells to facilitate foreign gene expression. However, the molecular mechanisms underlying Ca 2+ mediation of competent cell formation and identification of the key genes involved in the process remain unclear. In previous studies, the combined analysis of transcriptomics and proteomics revealed bhsA as one of the crucial genes. The gene ontology functional annotation of bhsA identified it as a member of the YhcN family encoding an outer membrane protein that confers resistance to various stresses. The IPR0108542 domain found within the protein plays a significant role in stress response and biofilm formation in E. coli. Analysis of the STRING database showed that the proteins interacting with bhsA are primarily involved in biofilm formation and stress resistance. Using the RED homologous recombination method, a bhsA deletion strain was constructed to verify its role in E. coli genetic transformation. Although the mutant strain showed no significant differences in morphology or growth trend when compared to the wild-type strain, its transformation efficiency decreased by 1.14- and 1.64-fold with plasmids pUC19 and pET-32a. Furthermore, the 1-N-phenylnaphthylamine assay indicated a 1.71-fold reduction in cell membrane permeability in the mutant strain., (© 2023 Wiley-VCH GmbH.)

Background: Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism. Results: We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA. Conclusions: Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs. [ABSTRACT FROM AUTHOR]

Abstract An efficient electrotransformation system that includes electrocompetent cells is a critical component for the success of large‐scale gene transduction and replication. The conditions of TG1 competent cell preparation and optimal electrotransformation were evaluated by investigating different parameters. Certain parameters for preparation of TG1 competent cells (≥8 × 1010 colony forming units (cfu)/μg DNA) include optimum culture time of monoclonal bacteria (8–10 hr), amplification growth concentration (approximately OD600 = 0.45), and culture volume (400 ml in 2 L conical flask). With increased storage of competent cells at −80°C, electrotransformation efficiency gradually decreased, but it remains greater than ≥ 1010 cfu/μg DNA 3 months later. Moreover, the recovery time of electrotransformation also influenced electrotransformation efficiency (1.5–2 hr for optimization). The optimized transformation efficiency of TG1 (≥8 × 1010 cfu/μg DNA) was observed under suitable electric voltage (2.5 kV), electric intensity (15 kV/cm), and electric time (3.5 ms) of electricity for plasmid transformation. Optimized DNA amount (0.01–100 ng) dissolved in water led to the high efficiency of plasmid transformation (≥8 × 1010 cfu/μg DNA), but had low efficiency when dissolved in T4 ligation buffer (≤3 × 1010 cfu/μg DNA). These results indicated that an optimized TG1 transformation system is useful for high electrotransformation efficiency under general laboratory conditions. The optimized TG1 transformation system might facilitate large‐scale gene transduction for phage display library construction.

Abstract Background Lactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating Lactobacillus reuteri ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency. Results Strains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A (trgA). To evaluate the role of trgA in transformation of DNA, we generated a trgA null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that trgA significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by trgA. Conclusions Our findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting trgA as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.

Abstract Background Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efficient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes-mediated root transformation of chickpea. Results Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fluorescent protein further confirmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efficiency. In addition, root transformation was found to be genotype-independent and the efficacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not affect functional studies involving infection of roots by fungal pathogen. Conclusions Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3–4 weeks, providing sufficient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.

Abstract Background Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR–Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. Results Here, we developed a RecET-assisted CRISPR–Cas9 genome editing method using a chromosome-borne Cas9–RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating l-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species. Conclusions The RecET-assisted CRISPR–Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable biomass using Corynebacterium species as cell factories.

Key message: An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5–9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species. [ABSTRACT FROM AUTHOR]

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One of small accessory genes between pol and env is tat gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing Escherichia coli (E. coli) in advance using adopted methods of M1 (MgCl2 + CaCl2) and M2 (CaCl2 + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic tat gene encoding protein JTAT was previously cloned into pBT-hisC. Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD600 = 4), incubated for 3 h. Pellets were harvested by centrifugation (4000 rpm; 4 °C; 15 min). Buffer B (10 mM Immidazole) was added into pellets, lysed by freeze-thaw followed by sonication. Supernatant was collected by centrifugation (10,000 rpm; 4 °C; 20 min) and purified using Ni-NTA Agarose resin, released by elution buffer (E) containing 400 mM Immidazole to collect purified protein twice (E1, E2). The protein was characterized by SDS-PAGE and Western Blot (WB), quantified (at λ595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 106) than M1 (3.10 × 105). The JTAT protein was expressed at a right size of 11.8 kDa. Concentration of 200 µM IPTG produced a significantly better protein yield (1.500 ± 0.089 mg/ml; P

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog (MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD600 = 0.8–1.0 and the co-cultivation at 25 °C for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gusA gene by PCR using the gusA specific primers in the T0 lines, there were 4 transgenic lines from 286 T0 lines (1.4% transformation efficiency). However, histochemical glucuronidase (GUS) assay demonstrated that only three of four transgenic lines exhibited gusA expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

Importance: The obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis , the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis , the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus is a clinically important pathogen that threatens human health due to its strong pathogenicity and drug resistance, leading to meningitis, endocarditis, and skin and soft tissue infections. Genetic manipulation in S. aureus is a powerful approach for characterizing the molecular mechanisms of bacterial drug resistance, pathogenicity, and virulence. However, a strong restriction barrier presents a major obstacle to the extensive utilization of genetic manipulation tools in clinical isolates of S. aureus . Here, we constructed a restriction-modification (RM) system silent CRISPR-Cas9 toolkit that synonymously eliminated the type I RM targets of S. aureus from plasmids, downsized plasmids using minicircle technology, and combined with a plasmid artificial modification (PAM) method to circumvent the type II RM system. The RM-silent CRISPR-Cas9 toolkit enables a significant improvement in transformation (10 5 -10 6 transformants per microgram plasmid in strains we tested) and high-success efficiency editing for gene deletion (knockout strain obtained in one-round electroporation) in a wide range of S. aureus species including clinical isolates of unknown genetic background. The RM-silent CRISPR-Cas9 toolkits could expedite the process of mutant construction in most S. aureus strains, and this approach could be applied to the design of other genetic toolkit plasmids for utilization in a wider range of S. aureus strains.

This paper compares several commonly used evaluation methods for the transformation efficiency of resource-based coastal cities, uses AHP and DEA analysis to analyze the main factors affecting the transformation efficiency, selects the statistical data of two coastal cities from 2013 to 2016, quantifies and weights each index, establishes a multidimensional, multi-level evaluation system combining qualitative and quantitative evaluation, analyzes the impact of different indexes on the transformation efficiency, and analyzes the internal motivation of transformation success according to the change trend of the transformation efficiency of the two cities. And the transformation efficiency of resource-based cities is evaluated. [ABSTRACT FROM AUTHOR]

Here, a rapid and easy transformation by electroporation technique for gene transfer in plants using cell penetrating amino nanocomplex (nanoplex) has been demonstrated in Nicotiana. Nanoplex was prepared using cell penetrating amino acids (CPAs) such as poly‐L‐lysine (PLL) and Argenine (Arg), in combination with the gold nanoparticles (AuNPs). PLLs‐modified nanoplex with zeta potential of 34.2 ± 1.22 mV charge showed 63.3% efficiency for gene transformation in plant cells as compared to 60% when modified with Arg and the zeta potential was found to be 30.0 ± 0.83 mV; whereas, the transformation efficiency without nanoplex was found to be 6.6%. The findings indicate that the zeta potential of positively charged nanocomplex (AuNPs/CPAs/DNA/CPAs) increases the transformation efficiency because of their ability to protect the DNA from electroporation wave and endogenous enzyme damage. Transformation was confirmed by GUS assay and amplification of npt gene. This technique may open up new possibilities of gene transfer in plants, which will enable to produce large number of transgenic plants. [ABSTRACT FROM AUTHOR]

Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae , Mycoplasma feriruminatoris , Mycoplasma gallisepticum and Mycoplasma pneumoniae , selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum , M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae , basing our study on more than 199,000 genome insertions. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is a model organism for Gram‐positive bacteria and widely used in the study of cellular functions and processes including protein secretion, sporulation, and signal transduction. It is also an important industrial host for the production of proteins and chemicals. Generally, genome editing of B. subtilis often needs the construction of integration vectors in Escherichia coli, linearizing the constructed plasmids, and subsequent transformation of the linear deoxyribonucleic acid via natural competence or electroporation. In this work, we examined the feasibility to directly transform and integrate B. subtilis using linear deoxyribonucleic acid from Gibson assembly without the need for cloning in E. coli. Linear deoxyribonucleic acid of 8–10 kb showed the highest transformation efficiency which was similar to that of using linearized plasmids constructed in E. coli. This method shortens the overall process from 1 week to 1 day and allows the integration of multiple genes in one step, providing a simple and fast method for genome editing in B. subtilis. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method. [ABSTRACT FROM AUTHOR]

In the Agrobacterium tumefaciens-mediated transformations of maize immature embryos (IEs), the common co-culturing media used are MS or N6-based (MC). Here, we used a novel co-culturing method in which maize 'Qi319' IEs inoculated with Agrobacterium-harboring target vector were placed on dry filter paper (DC) in a petri dish. To compare the effects of the DC and MC co-culturing methods on transformation efficiency, we designed three experiments: (1) A. tumefaciens strain AGL1 independently carrying two plasmids, pXQD12 and pXQD70; (2) two A. tumefaciens strains, AGL1 and EHA105, carrying pXQD12; and (3) strains AGL1 and EHA105 each independently inoculated with pXQD12 and pXQD70 for different infiltration periods, 5, 10, 15, 20 and 25 min. We used A. tumefaciens to inoculate IEs derived from maize ears 9-15 d after pollination, and then IEs were placed in petri dishes for co-culturing. The DC treatment significantly increased the percentage of IEs expressing green fluorescence protein (%GFP), indicating positive transformants. DC-treated IEs had ~ 3 to 4 times the %GFP compared with MC-treated IEs at 8 d after inoculation (3 d co-culture and 5 d restoration). The average regeneration frequency (%GFP positive regenerated calli of infected IEs) and stable transformation frequency (%GFP positive T0 plants of infected IEs) significantly increased with the DC treatment. Thus, the DC method may be used to develop a more efficient Agrobacterium-mediated transformation method for maize IEs. [ABSTRACT FROM AUTHOR]

Summary: Populus alba is widely distributed and cultivated in Europe and Asia. This species has been used for diverse studies. In this study, we assembled a de novo genome sequence of P. alba var. pyramidalis (= P. bolleana) and confirmed its high transformation efficiency and short transformation time by experiments. Through a process of hybrid genome assembly, a total of 464 M of the genome was assembled. Annotation analyses predicted 37 901 protein‐coding genes. This genome is highly collinear to that of P. trichocarpa, with most genes having orthologs in the two species. We found a marked expansion of gene families related to histone and the hormone auxin but loss of disease resistance genes in P. alba if compared with the closely related P. trichocarpa. The genome sequence presented here represents a valuable resource for further molecular functional analyses of this species as a new tree model, poplar breeding practices and comparative genomic analyses across different poplars. [ABSTRACT FROM AUTHOR]