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

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

Frontiers in Nanotechnology ◽

10.3389/fnano.2021.777810 ◽

2021 ◽

Vol 3 ◽

Author(s):

Asmita Devkota ◽

Anju Pandey ◽

Zeinab Yadegari ◽

Korsi Dumenyo ◽

Ali Taheri

Keyword(s):

Incubation Time ◽

Plasmid Dna ◽

Carbon Dots ◽

Dna Delivery ◽

Bacterial Cells ◽

Bacterial Strains ◽

E Coli ◽

Glucose Carbon ◽

Time Required ◽

Plasmid Size

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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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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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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Evaluation of biocompatibility and efficiency of plasmid DNA delivery by gene-activated hydrogels in vitro

Genes and Cells ◽

10.23868/201906017 ◽

2019 ◽

Vol XIV (2) ◽

Author(s):

I.Y. Bozo ◽

A.A. Titova ◽

M.N. Zhuravleva ◽

A.I. Bilyalov ◽

M.O. Mavlikeev ◽

...

Keyword(s):

Plasmid Dna ◽

Dna Delivery

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Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-67-72 ◽

2019 ◽

Vol 35 (6) ◽

pp. 67-72 ◽

Cited By ~ 1

Author(s):

I.V. Manukhov ◽

L.S. Yaguzhinsky ◽

M.V. Bermeshev ◽

M.A. Zisman ◽

V.G. Pevgov ◽

...

Keyword(s):

Toxic Effect ◽

Atmospheric Oxygen ◽

Inducible Promoter ◽

Oxygen Species ◽

Bacterial Cells ◽

Unique Identifier ◽

E Coli ◽

Lux Biosensors ◽

High Level ◽

Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

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