Glucosamine/β-Alanine Carbon Dots Use as DNA Carriers Into E. coli Cells

Introducing foreign DNA into bacterial cells is essential in functional genomics and molecular research. Currently, heat shock and electroporation are the two major techniques of gene delivery in bacterial cells. However, both the techniques are time and resource consuming and are limited to a few species or strains of bacteria and there is a need to develop new transformation alternatives. Carbon dots with unique features such as facile synthesis, ease of functionalization, nontoxicity, and biocompatibility are considered novel biomolecule nanocarriers. In this study, we synthesized and evaluated DNA delivery potential of four carbon dots including: 1) amine-coated carbon dots (NH2-FCDs); 2) carboxylate carbon dots (COOH-FCDs); 3) L-arginine and glucose carbon dots (N-CDs), and 4) citric acid and polyethyleneimine (PEI) carbon dots into Escherichia. coli cells. We evaluated the minimum incubation time required for the plasmid DNA delivery and the maximum plasmid size that can be delivered into E. coli cells using these CDs. Bacteria were incubated with carbon dots solution for different lengths of time and plated on selection media. Transformed colonies were counted and data were analyzed to identify the optimum incubation time and measure DNA delivery of these CDs with plasmids of different sizes. Our study demonstrated that among all these CDs, only carboxylate carbon dots (COOH-FCDs) prepared from glucosamine and β-alanine were able to deliver plasmid DNA into E. coli cells and the best incubation time was between 30 and 60 min. The maximum plasmid size that could be delivered using these CDs was approximately 10 kb and transformation efficiency decreased with larger plasmids. This study shows the capacity of COOH-CDs to deliver plasmid DNA into bacteria with an immense potential to combine with modern genome-editing tools. However, further studies are needed to evaluate their potential in DNA delivery in other bacterial strains.

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Citric acid/β-alanine carbon dots as a novel tool for delivery of plasmid DNA into E. coli cells

Scientific Reports ◽

10.1038/s41598-021-03437-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anju Pandey ◽

Asmita Devkota ◽

Anil Sigdel ◽

Zeinab Yadegari ◽

Korsi Dumenyo ◽

...

Keyword(s):

Citric Acid ◽

Plasmid Dna ◽

Carbon Dots ◽

Dna Delivery ◽

Resistant Organism ◽

Bacterial Cells ◽

Bacterial Membranes ◽

E Coli ◽

Foreign Dna ◽

Repulsive Forces

AbstractSuccessful delivery of plasmid DNA into the microbial cells is fundamental in recombinant DNA technology. Natural bacterial transformation is limited to only certain species due in part to the repulsive forces between negatively charged DNA and bacterial membranes. Most common method of DNA delivery into bacteria is artificial transformation through heat shock and electroporation. These methods require sophisticated instruments and tedious steps in preparation of competent cells. Transformation by conjugation is also not applicable to all plasmids. Nanoparticles have been used successfully in therapeutics for drug delivery into animal cells. They are starting to gain popularity in plant sciences as novel DNA nano carriers. Despite their promise as tool for DNA delivery, their use in microbial cell transformation has not been reported yet. Here we report the synthesis of carbon dots (CDs) from citric acid and β-alanine and their use in DNA delivery into E. coli cells. CDs were fabricated using microwave assisted synthesis. Plasmids carrying RFP reporter and ampicillin resistance genes were transferred to bacterial cells and further confirmed using polymerase chain reaction. Our findings indicate that CDs can be used successfully for delivery of foreign DNA of up to 10 kb into E. coli. We have demonstrated the use of β-alanine/citric acid carbon dots as nanocarriers of DNA into E. coli cells and identified their limitation in terms of the size of plasmid DNA they could carry. Use of these carbon dots is a novel method in foreign DNA delivery into bacterial cells and have a potential for the transformation of resistant organism for which there is still no reliable DNA delivery systems.

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One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot101212 ◽

2021 ◽

Vol 2021 (11) ◽

pp. pdb.prot101212 ◽

Cited By ~ 1

Author(s):

Michael R. Green ◽

Joseph Sambrook

Keyword(s):

Escherichia Coli ◽

Convenient Method ◽

Plasmid Dna ◽

Transformation Efficiency ◽

Bacterial Cells ◽

E Coli ◽

One Step ◽

And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

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Utilization of Mn-Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus

Advances in Materials Science and Engineering ◽

10.1155/2020/9718706 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Thi-Diem Bui ◽

Quang-Liem Nguyen ◽

Thi-Bich Luong ◽

Van Thuan Le ◽

Van-Dat Doan

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Quantum Dots ◽

Fluorescent Labeling ◽

Core Shell ◽

Bacterial Cells ◽

Bacterial Strains ◽

Methicillin Resistant ◽

E Coli ◽

Mn Doped

In this study, Mn-doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using polyethylene glycol as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti-E. coli antibody and anti-MRSA antibody via protein A supported by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The detection was performed for the bacterial strains cultivated in Luria-Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

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Photon-Induced Superior Antibacterial Activity of Palladium-Decorated, Magnetically Separable Fe3O4/Pd/mpg-C3N4 Nanocomposites

Molecules ◽

10.3390/molecules24213888 ◽

2019 ◽

Vol 24 (21) ◽

pp. 3888 ◽

Cited By ~ 1

Author(s):

Deepika Thakur ◽

Qui Thanh Hoai Ta ◽

Jin-Seo Noh

Keyword(s):

Antibacterial Activity ◽

Solution Method ◽

Pd Nanoparticles ◽

Bacterial Degradation ◽

Photoluminescence Intensity ◽

Bacterial Cells ◽

Bacterial Strains ◽

E Coli ◽

Carrier Recombination ◽

Solar Light Irradiation

Three-component nanocomposites (Fe3O4/Pd/mpg-C3N4) have been systematically synthesized using a three-step solution method for the photocatalytic bacterial decontamination. The mesoporous g-C3N4 nanosheets (mpg-C3N4), which were prepared by the acid treatment, showed a great improvement in photocatalytic performance. The photoluminescence intensity of the mpg-C3N4 nanosheets was disclosed to drop about 60% from the value of normal g-C3N4 nanosheets. Decoration of mpg-C3N4 with palladium (Pd) nanoparticles led to the effective suppression of carrier recombination and the carrier migration to Fe3O4 nanoparticles. It was revealed that the three-component nanocomposites degraded 99.9% of E. coli and 99.8% of S. aureus bacterial strains within 2 h of solar light irradiation at a 100 μg/mL concentration, demonstrating their superb photocatalytic antibacterial activity. In addition, the nanocomposites could be easily separated from the bacterial cells and repeatedly used for photocatalytic bacterial degradation with good recyclability. The strong photon-induced antibacterial activity and good recyclability of the three-component nanocomposites may represent their potential as a promising antibacterial photocatalyst.

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Trans-Kingdom Conjugation Within Solid Media from Escherichia coli to Saccharomyces cerevisiae

10.20944/preprints201909.0340.v1 ◽

2019 ◽

Author(s):

Maximillian P.M. Soltysiak ◽

Rebecca S. Meaney ◽

Samir Hamadache ◽

Preetam Janakirama ◽

David R. Edgell ◽

...

Keyword(s):

Donor Cell ◽

Dna Delivery ◽

Dna Transfer ◽

Liquid Media ◽

E Coli ◽

Solid Media ◽

Wide Range ◽

Semi Solid ◽

Plasmid Size ◽

Synthesis Protocol

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from E. coli to S. cerevisiae. We demonstrated that there was no substantial decrease in conjugation frequency as plasmid size increased—up to 138.5 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids

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When Beneficial Biofilm on Materials Is Needed: Electrostatic Attachment of Living Bacterial Cells Induces Biofilm Formation

Frontiers in Materials ◽

10.3389/fmats.2021.624631 ◽

2021 ◽

Vol 8 ◽

Author(s):

Dmitrii Deev ◽

Iaroslav Rybkin ◽

Tomaž Rijavec ◽

Aleš Lapanje

Keyword(s):

Biofilm Formation ◽

Bacterial Attachment ◽

Wall Structure ◽

Bacterial Cells ◽

Bacterial Strains ◽

Microbial Composition ◽

E Coli ◽

Environmental Bacteria ◽

Artificial Biofilm ◽

Biofilm Prevention

Bacterial attachment is crucial in many biotechnological applications, but many important bacterial strains cannot form biofilms. Biofilms can damage materials, and current strategies to manage biofilms are focused on inhibition and removal of biofilm. Biofilm formation is inevitable when materials are exposed to microbes and instead of biofilm prevention, we propose management of microbial composition by formation of biofilms with beneficial microbes. Since bacteria need to overcome a high repulsive force to attach to the surface and later to grow and multiply on it, electrostatic modification of the surfaces of cells or the material by polyelectrolytes (PE) was used in our approach, enabling efficient attachment of viable bacterial cells. Since highly positively charged PEs are known to be bactericidal, they were acetylated to reduce their toxicity, while preserving their net positive charge and ensuring cell viability. In our study bacterial strains were selected according to their intrinsic capability of biofilm formation, their shape variety and cell wall structure. These strains were tested to compare how the artificially prepared vs. natural biofilms can be used to populate the surface with beneficial bacteria. Using an artificial biofilm constructed of the potentially probiotic isolate Bacillus sp. strain 25.2. M, reduced the attachment and induced complete inhibition of E. coli growth over the biofilm. This study also revealed that the modification of the surfaces of cells or material by polyelectrolytes allows the deposition of bacterial cells, biofilm formation and attachment of biofilm non-forming cells onto surfaces. In this way, artificial biofilms with extended stability can be constructed, leading to selective pressure on further colonization of environmental bacteria.

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Antibacterial Effect of Plant Resin Collected from Tetrigona apicalis (Smith, 1857) in Thung Salaeng Luang National Park, Phitsanulok

Walailak Journal of Science and Technology (WJST) ◽

10.48048/wjst.2018.4596 ◽

2017 ◽

Vol 15 (8) ◽

pp. 599-607

Author(s):

Sathirapong KRAIKONGJIT ◽

Touchkanin JONGJITVIMOL ◽

Naklao MIANJINDA ◽

Nutta SIRITHEP ◽

Thodsaporn KAEWBOR ◽

...

Keyword(s):

Phenolic Compounds ◽

National Park ◽

Stingless Bees ◽

Antibacterial Properties ◽

Total Phenolic ◽

Bacterial Cells ◽

Bacterial Strains ◽

E Coli ◽

Phenolic Contents ◽

Two Samples

Tetrigona apicalis (Smith, 1857) is a common species of stingless bee found in lower northern Thailand. In previous studies, the propolis of stingless bees has been shown to have antibacterial properties, due to its chemically contained phenolic contents. The major component of propolis is resin. The purpose of this study, therefore, was to evaluate the antibacterial activities of crude resin extracts by disk diffusion and broth microdilution methods. We also determined the total phenolic contents using the Folin-Ciocalteau method and, to detect individual polyphenolic contents, we used the high performance liquid chromatographic method. Two samples of resin were collected from Thung Salaeng Luang National Park, Phitsanulok. The first sample was from fresh plants, which stingless bees used for nest construction. The second sample was taken from entrances of the bee’s nest. All samples were macerated in 30 % ethanol and incubated at room temperature for 14 days. The supernatants were filtered and ethanol residues then removed as ethanolic resin extracts (eREs). The antibacterial activity of the extracted resins against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 was examined. The disks containing 9 and 14 mg of eREs produced obvious inhibition zones against S. aureus, but did not show zones against E. coli and P. aeruginosa. The minimum inhibitory concentrations (MICs) of the eREs against the bacterial strains tested were variously between 6 and 48 mg/ml, whereas the minimum bactericidal concentrations (MBCs) were from 12 to 48 mg/ml. The amount of the total phenolic compounds in the eREs from the fresh resin was 9,908 mg of pyrogallol equivalent (PGE) per kg of eREs, and from the nest entrances, 14,740 mg per kg. We also found that hydroquinin had the highest concentration in both extracts. In conclusion, the crude resin extracts demonstrated antibacterial properties against the S. aureus, E. coli, and P. aeruginosa strains tested. They also contained phenolic compounds which were active antibacterial agents. We have identified new and novel knowledge which can be used as preliminary data, leading to further, more detailed, investigation of the mechanistic action of the resin against bacterial cells.

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Antibacterial Properties of Citric Acid/β-Alanine Carbon Dots against Gram-Negative Bacteria

Nanomaterials ◽

10.3390/nano11082012 ◽

2021 ◽

Vol 11 (8) ◽

pp. 2012

Author(s):

Anju Pandey ◽

Asmita Devkota ◽

Zeinab Yadegari ◽

Korsi Dumenyo ◽

Ali Taheri

Keyword(s):

Citric Acid ◽

Incubation Time ◽

Carbon Dots ◽

Bacterial Species ◽

Synthesis Process ◽

Diffusion Method ◽

Multi Drug Resistance ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli

While multi-drug resistance in bacteria is an emerging concern in public health, using carbon dots (CDs) as a new source of antimicrobial activity is gaining popularity due to their antimicrobial and non-toxic properties. Here we prepared carbon dots from citric acid and β-alanine and demonstrated their ability to inhibit the growth of diverse groups of Gram-negative bacteria, including E. coli, Salmonella, Pseudomonas, Agrobacterium, and Pectobacterium species. Carbon dots were prepared using a one-pot, three-minute synthesis process in a commercial microwave oven (700 W). The antibacterial activity of these CDs was studied using the well-diffusion method, and their minimal inhibitory concentration was determined by exposing bacterial cells for 20 h to different concentrations of CDs ranging from 0.5 to 10 mg/mL. Our finding indicates that these CDs can be an effective alternative to commercially available antibiotics. We also demonstrated the minimum incubation time required for complete inhibition of bacterial growth, which varied depending on bacterial species. With 15-min incubation time, A. tumefaciens and P. aeruginosa were the most sensitive strains, whereas E. coli and S. enterica were the most resistant bacterial strains requiring over 20 h incubation with CDs.

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Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens

Journal of Clinical Microbiology ◽

10.1128/jcm.36.3.662-668.1998 ◽

1998 ◽

Vol 36 (3) ◽

pp. 662-668 ◽

Cited By ~ 179

Author(s):

Jeannette Adu-Bobie ◽

Gad Frankel ◽

Christopher Bain ◽

Azizedite Guedes Goncalves ◽

Luiz R. Trabulsi ◽

...

Keyword(s):

Outer Membrane Proteins ◽

Pcr Primers ◽

Eukaryotic Cell ◽

Polyclonal Antiserum ◽

Microbial Pathogens ◽

Enzyme Linked Immunosorbent Assay ◽

Bacterial Cells ◽

Bacterial Strains ◽

E Coli ◽

Immunofluorescence Labelling

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenicEscherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), andCitrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.

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Antibacterial Activity of cLFchimera and its Synergistic Potential with Antibiotics against Some Foodborne Pathogens Bacteria

10.21203/rs.3.rs-31015/v1 ◽

2020 ◽

Author(s):

Sahar Roshanak ◽

Zana Pirkhezranian ◽

Fakhri Shahidi ◽

Mohammad hadi Sekhavati

Keyword(s):

Foodborne Pathogens ◽

Synergistic Effects ◽

Salmonella Typhi ◽

Synergistic Activity ◽

Bacterial Cells ◽

Bacterial Strains ◽

E Coli ◽

Viable Cells ◽

Electron Microscopy Analysis ◽

Microscopy Analysis

Abstract Background: Frequent and unlimited use of antibiotics caused the development of antibiotic resistance by microorganisms. Therefore, there is an argent need to discover novel antibacterial agents or a combination of agents as a safe treatment strategy for various infections. The aim of the present study was to evaluate the synergistic effects of cLFchimera, an antimicrobial peptides (AMPs), and antibiotics on several foodborne bacterial strains. Methods: A checkerboard method was used to determine the synergistic effects of cLFchimera and several antibiotics (Gentamicin, Cefazolin and Ceftazidime) on bacterial strains (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi). Results: The combination of cLFchimera and antibiotics generated a total and partial synergistic interaction for all foodborne bacterial strain used in the present study (FIC= 0.25 to 0.77). In most cases, the effect of peptide and antibiotic synergist on release of cellular content was not different compared to antibiotics when they used alone, but the count of viable cells significantly decreased in combination peptide and antibiotics treatments. Generally, antibacterial dynamics of the combination of peptide and antibiotics showed an increase and stable trend after reaching the peak point for E. coli, P. aeruginosa and S. typhi, respectively. Scanning electron microscopy analysis showed that bacterial cells treated with the combination Gentamycin and cLFchimera were markedly damaged, and most of the outermost layer of the bacterial cells disappeared. Conclusion: Overall, our results may suggest that cLFchimera mediated its synergistic activity independent to antibiotics mode of action by disrupting the cell membrane and intramolecular mechanisms.

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