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.
Highlights
Genetic engineering in prokaryotic unicellular organisms has reached newer heights with metabolic and pathway engineering, sometimes leading to drastic change in the identity of the parent strain [1,2]
There has been a need for specific transformation methods for each genome and nucleus transformation has been the most difficult to achieve because of the resistance of two membranes, cell wall/membrane followed by nuclear membrane [6,7]
The rupture of the tough algal cell wall and the aforesaid membrane followed by successful integration and above all, survival of the cell to generate transformed progeny is a delicate exercise, which points to the handful of protocols with low efficiencies and high sophistication [8,9]
Summary
Genetic engineering in prokaryotic unicellular organisms has reached newer heights with metabolic and pathway engineering, sometimes leading to drastic change in the identity of the parent strain [1,2]. Eukaryotic unicellular organisms like microalgae pose several bottlenecks to genetic engineering, foremost among them is transformation [3,4,5]. There has been a need for specific transformation methods for each genome and nucleus transformation has been the most difficult to achieve because of the resistance of two membranes, cell wall/membrane followed by nuclear membrane [6,7]. In spite of such efforts, genetic engineering in eukaryotic microalgae is not well-established, compared with bacterial transformation [2,5,7,11,12,13]. The development of simpler and more efficient methodology is needed
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
