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

Asmita Devkota,Korsi Dumenyo,Zeinab Yadegari,Ali Taheri,Anju Pandey

doi:10.3389/fnano.2021.777810

Asmita Devkota, Korsi Dumenyo + Show 3 more

Open Access

https://doi.org/10.3389/fnano.2021.777810

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Abstract

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.

Highlights

Gene delivery and DNA manipulation are essential in most of the functional genomics research and molecular activities including cloning, mutant generation, and construction of DNA libraries
Four types of carbon dots (CDs) were synthesized from different precursors and their physical properties and gene delivery competencies for individual plasmid sizes were studied at distinct incubation times
In the COOH-FCDs, distinct FTIR peaks were obtained at 3,340 cm−1 indicative of various amino and alcohol groups (Supplementary Figure S2)

Summary

Introduction

Gene delivery and DNA manipulation are essential in most of the functional genomics research and molecular activities including cloning, mutant generation, and construction of DNA libraries. The most commonly used artificial transformation methods in bacteria are: 1) heat shock method that incorporates CaCl2 treatment of bacterial cells and 2) electroporation (Divya et al, 2011) Both these methods have drawbacks such as lengthy procedures in preparing the competent cells or the need for sophisticated instruments to generate electric shock. Xu et al (2004) reported the synthesis of CDs for the first time while working on the purification of single-walled carbon nanotubes (SWCNT). Soon after this discovery, many scientists were attracted to carbon dots, which have similar functions to metal-based nanomaterials but are less toxic to the mammalian cells and the environment. Dispersibility of CDs is the result of surface functional groups whereas their toxicity is mainly due to their core region (Huang et al, 2015; Kim et al, 2018; Yao et al, 2019)

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