Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

Highlights

• Mg aminoclays were synthesized via a simple sol-gel process and characterized.

• They are arranged in layered sheets of structure R₈Si₈Mg₆O₁₆(OH)₄ where R = CH₂CH₂NH₂.

• Non-competent E. coli JM109 was genetically modified using cationic Mg aminoclays.

• Maximum transformation efficiency achieved was 7.0 × 10³ CFU/μg pUC19.

• Cost-effective, rapid, convenient and risk-free optimized transformation solution.

Abstract

Magnesium aminoclays were synthesized and used to transform non-competent Escherichia coli JM109 using the exogenous plasmid pUC19. The structure determined for the Mg aminoclays is analogous to 2:1 trioctahedral smectites such as talc, with an approximate composition R₈Si₈Mg₆O₁₆(OH)₄, where R = CH₂CH₂NH₂, morphologically arranged in layered sheets. Mg aminoclays were employed as a cationic vehicle that enabled the passage of plasmids across the cell envelope and led to genetic modification of the host. A stock solution of 10 mg/mL of Mg aminoclays was prepared, mixed with E. coli JM109 and pUC19 plasmid, and spread over Petri dishes containing lysogeny broth (LB), isopropyl β-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (1–4%). The transformation efficiency obtained was higher for 1% and 2% agar even though transformation also occurred at agar concentrations of 3% and 4%. The optical density of E. coli JM109 and spreading time were also adjusted, favoring transformation when cells were used in their exponential growth phase (OD₆₀₀ = 1.0) and spread for 90 s. Transformation was confirmed by the growth of blue colonies in LB/IPTG/X-gal/agar Petri dishes containing ampicillin, by regrowth of biomass in liquid media containing ampicillin and by agarose gel electrophoresis of the linearized pUC19 plasmid that followed plasmidic DNA extraction from 4 blue colonies. The maximum transformation efficiency achieved was 7.0 × 10³ CFU/μg pUC19. This transformation approach proved to be suitable for a convenient, cost-effective, room-temperature, risk-free and rapid transformation of non-competent E. coli JM109.

Introduction

Genetic modification or transformation of microorganisms, perceived as a consequence of an intake of external DNA with the purpose of originating cells capable of producing interesting compounds for the pharmaceutical, food or energy sectors, is a fundamental process for promoting advances in biotechnology and is still predominantly accomplished through heat-shock or electroporation. However, these techniques share the disadvantage of requiring the preparation of competent cells causing them to become inconvenient and time-consuming procedures. Electroporation in particular has the added inevitability of manipulation of cuvettes that must be submitted to dangerous high-voltages and are ephemeral expensive consumables. Even in more convenient cases contemplating the acquisition or freeze-storage of competent cells that can efficiently be transformed with a specific plasmid by these techniques, transformation using other plasmids is not guaranteed. Furthermore, several species of bacteria and yeast with great potential for biotechnology applications are completely recalcitrant to any lab-induced genetic modification through these or any other of the less frequent transformation methods available (Ren et al., 2019). Therefore, efficient emergent alternative transformation methods and their optimization resulting in routine laboratory protocols are crucial to unravel the potential of recombinant DNA technology which stimulates the development of the field of biotechnology. Novel delivery agents have recently been developed such as a cationic fusion peptide that penetrates Escherichia coli enabling the expression of a large-sized plasmid (pMSR227, 205 kb) (Islam et al., 2019) and arginine-glucose functionalized hydroxyapatite nanoparticles, that can be used for transformation of at least one Gram-negative and one Gram-positive bacterial species (Deshmukh et al., 2019a) and one species of yeast (Deshmukh et al., 2019b) with high transformation efficiencies.

Advances in the application of synthetic nanostructured materials produced by wet-chemistry strategies have recently been accomplished in various fields such as photocatalysis (Morassaei et al., 2017; Zinatloo-Ajabshir and Salavati-Niasari, 2017), energy storage (Ghodrati et al., 2020; Mousavi-Kamazani et al., 2020) or selective catalytic reduction (SCR) (Zinatloo-Ajabshir et al., 2021). In particular, wet-chemistry has been used to produce clays containing organic groups which have been investigated with great detail during the last couple of decades due to their potential use in smart materials (Zhuk et al., 2011), batteries (Zhang et al., 2008), polymer engineering (Ruiz-Hitzky et al., 2005), protective barriers (Yao et al., 2012), nanocomposites, (Balazs et al., 2006; Podsiadlo et al., 2007; Bonderer et al., 2008; Munch et al., 2008; Priolo et al., 2010), immobilization of catalysts (Miao et al., 2006; Scheuermann et al., 2009), purification of mixtures (Ding and Henrichs, 2002; Hsu et al., 2010), tissue engineering (Pappas et al., 2007; Liu et al., 2013, Liu et al., 2014), targeted drug delivery (Viseras et al., 2010) and several other biological and environmental applications (Bui et al., 2018). The synthesis of functional clays capable of specific interaction with cells, molecules or substrates is expected to be advantageous for many new in vivo applications. Consequently, a new class of synthetic clays with organic-inorganic layers derivatives of 2:1 trioctahedral phyllosilicates has been developed, possessing amino functionalized propyl groups occupying the interlayer locations (Burkett et al., 1997; Mann et al., 1997). These aminoclays can be prepared through a simple protocol using an organosilane precursor and magnesium or calcium chloride at room-temperature through a sol-gel process. Nanobiohybrid inorganic compounds can be easy prepared using aminoclays by incorporating functional biomolecules, bearing potential for applications as biocatalysts (Lee et al., 2013), biosensors (Mann, 2009; Mousty, 2010) or drug delivery nanocarriers (Patil et al., 2005; Patil and Mann, 2008; Chaturbedy et al., 2010). Additionally, the positive charges from the amino groups of the aminoclay nanoparticles at physiological pH values makes them excellent candidates for electrostatic binding with DNA (Whilton et al., 1998; Datta et al., 2013) and diffuse as a complex through an aqueous environment without substantial adverse aggregation. Certain clay mineral nanomaterials and particularly aminoclays have been reported as acting as a bactericide (Williams et al., 2011; Ito et al., 2018; Abhinayaa et al., 2019; Gaálová et al., 2019; Li et al., 2019). The bactericidal activity of aminoclays is a consequence of the easy adsorption of the aminoclays onto bacterial cell surfaces followed by penetration of the cell wall and membrane(s), leading to membrane damage, depolarization, leakage of intracellular components and cell death (Chandrasekaran et al., 2011), but cell survival can potentially be controlled by adjusting the concentration of aminoclays that the microorganisms are exposed. Aminoclays have also already been reported to have low toxicity while being effective as a cationic vehicle for enhancing adenovirus-mediated gene transfer in mammalian cells (Kim et al., 2017). Accordingly, delivery of plasmids bound to aminoclays to the interior of a vast range of different species of microorganisms can potentially be achieved using an aminoclay concentration under a threshold that ensures host survival and having the exogenous plasmid protected from restriction enzymes due to a self-assembled nanoarmor (Demanèche et al., 2001). Moreover, Mg aminoclays have already successfully led to the expression of plasmids in E. coli XL1-Blue and Streptococcus mutans (Choi et al., 2013) and in Paenibacillus riograndensis and Paenibacillus polymyxa (Brito et al., 2017), exhibiting potential to be used for transformation of other microorganisms that are recalcitrant to be modified genetically by conventional methods.

The main scope of this work was to synthesize Mg aminoclays via a simple sol-gel process, characterize and explore their use as a vehicle to deliver the plasmid pUC19 across the cell envelope of a non-competent version of a laboratory workhorse widely used as cloning host, Escherichia coli JM109, and refine different conditions towards achieving maximum transformation efficiency. The work thus allows to evaluate the potential of an alternative approach for bacterial transformation, which represents an easy and rapid solution that can prevail for many common laboratory needs. This work can also be used as reference for future transformation attempts on other microorganisms and stimulate the adoption of plasmid delivery agents as a strategy to transform recalcitrant species relevant to the field of biotechnology.