A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

Abstract

Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H2N(CH2)3]8Si8Mg6O12(OH)4), for nuclear transformation into eukaryotic microalgae. TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.03×102 transformants/µg DNA), second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.

Production of chemicals from microalgae lipids – status and perspectives

“…For the prominent green alga C. reinhardtii , the largest spectrum of DNA delivery techniques was developed (Fig. – 1–4) : Biolistics: Bombarding the cells with DNA‐coated tungsten or gold microparticles , Vortexing the cells in the presence of DNA and silicon carbide whiskers , glass beads , or amino clay nanoparticles , Electroporation: Applying a strong electrical field to the cells, Agrobacterium tumefaciens : Utilization of bacteria that actively inject DNA into the cells . …”

A Simple Method for Removal of the <b><i>Chlamydomonas reinhardtii</i></b> Cell Wall Using a Commercially Available Subtilisin (Alcalase)

“…The main issue to overcome in algal transformation is the complexity of cell wall structures [Kim et al, 2014]. The cell wall of Chlamydomonas is multilayered, including an extracellular coat composed of hydroxyprolinerich glycoproteins and cellulosic polysaccharides [Goodenough and Heuser, 1985].…”

“…The cell wall of Chlamydomonas is multilayered, including an extracellular coat composed of hydroxyprolinerich glycoproteins and cellulosic polysaccharides [Goodenough and Heuser, 1985]. An important challenge for microalgal transformation is the selective disruption of the cell membrane, without loss of cell function [Kim et al, 2014]. Therefore, we evaluated potential alternatives to the conventionally used Chlamydomonas cell wall lytic enzyme.…”

“…In the past decade, genetic transformation methods have been developed for both microand macroalgae [Greiner et al, 2017]. However, genetic transformation is still a challenge for some algae owing to issues with gene delivery and genetic divergence among algal species [Kim et al, 2014]. Recent research in the field has focused on CRISPR/Cas-based gene editing [Jiang et al, 2014;Shin et al, 2016], which achieves targeted gene editing using a protein-gRNA complex delivery method.…”

“…The glass bead method is inexpensive and effective for gene delivery; however, a cell wall-degrading enzyme, e.g., autolysin, is needed to increase transformation efficiency. More recently, the aminoclay-based transformation method has been developed; this method does not require cell wall removal but involves the preparation of positively charged nanoparticles [Kim et al, 2014].…”