preparation of electrocompetent agrobacterium tumefaciens culture for microalgal genetic engineering

Stable Gene Integration in Chlorella vulgaris Using Agrobacterium tumefaciens

Agrobacterium tumefaciens, a gram-negative soil-borne bacterium, possesses a unique interkingdom gene transfer ability, earning it the title &#34;natural genetic engineer&#34;1. This bacterium can transfer DNA (T-DNA) from a tumor-inducing plasmid (Ti-Plasmid) into host cells through a Type IV secretion system, resulting in the integration and expression of the T-DNA within the host genome1,2,3,4. In the natural setting, this process leads to tumor formation in plants, commonly known as crown gall disease. However, Agrobacterium can also transfer T-DNA into various other organisms, including yeast, fungi, algae, sea urchin embryos, and even human cells under laboratory conditions5,6,7,8.
Exploiting this natural system, Agrobacterium tumefaciens-mediated transformation (AMT) enables the random integration of gene(s) of interest into a host cell&#39;s nuclear genome by modifying the T-DNA region of the Ti-plasmid. For this purpose, a widely used AMT plant expression vector is pCAMBIA13029. Researchers can employ simple cloning workflows in E. coli before transferring the desired vector into A. tumefaciens for subsequent transfer to the host of interest.
Green microalgae are eukaryotes that share many similarities with land plants but are highly recalcitrant to genetic modification. However, genetic transformation plays a crucial role in both fundamental and biotechnological research of microalgae. In several microalgae species, particularly Chlamydomonas reinhardtii, genetic transformation via AMT has successfully introduced transgenes such as human interleukin-2 (hIL-2), the severe acute respiratory syndrome coronavirus 2 receptor-binding domain (SARS-CoV-2 RBD), and two antimicrobial peptides (AMPs)10,11,12,13. Among these, Chlorella vulgaris, a less fastidious and fast-growing green algae species, holds significant potential for the sustainable production of carbohydrates, proteins, nutraceuticals, pigments, and other high-value compounds14. However, the lack of reliable tools for creating transgenic strains of C. vulgaris hampers its commercial progress. Since there have been only a limited number of published works utilizing AMT in C. vulgaris15, and given the considerable differences between plant and microalgae cultivation, optimizing the AMT protocol becomes essential.
In this study, researchers inserted green fluorescent protein (mGFP5) downstream of the Cauliflower Mosaic Virus (CamV) 35S promoter and added a histidine tag to use it as a reporter gene for protein expression. Transformants were selected using Hygromycin B, and after subculturing for over twenty generations, the transformation remained stable. The pCAMBIA1302 plasmid employed in this work can be readily adapted to contain any gene of interest. Furthermore, the method and materials presented can be adjusted for other green algae species with an active CamV35S promoter, as this promoter is used for Hygromycin selection.

Author Spotlight: Optimized Transformation Protocol for Chlorella vulgaris Using Agrobacterium tumefaciens 

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

The scope of the research focused on the development and optimization of a protocol for the transformation of chlorella vulgaris. We aimed to create a stable transgenic strains of chlorella vulgaris using agrobacterium tumefaciens and a specific plasmid, pCAMBIA1302, that allowed them to insert gene of interest and the potential for bio technological applications. Metabolic engineering and synthetic biology, microscopy and imaging, CRISPR-Cas9 gene editing, bioinformatics, computational modeling, remote sensing and satellite imaging, nanotechnology, bioprocessing, and downstream processing.This study introduces an optimized agrobacterium tumefaciens media transformation protocol for the microalgae chlorella vulgaris using a specific plasmid and marker. It lays groundwork for enhancing microalgae by technology, overcoming previous limitations in reliable transformation tools for C.vulgaris.

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Preparation of Electrocompetent  Agrobacterium tumefaciens  Culture for Microalgal Genetic Engineering  

Preparation of Electrocompetent Agrobacterium tumefaciens Culture for Microalgal Genetic Engineering

Start by diluting 0.5 microliters of the overnight cultures of Agrobacterium tumefaciens culture with 50 milliliters of autoclaved ultrapure water. Spread 10 microliters of this diluted culture on a lysogeny broth or LB plate. Incubate the plate at 28 to 30 degrees Celsius for one to three days.Now, inoculate a single colony in 20 milliliters of LB media supplemented with rifampicin. Incubate the culture overnight at 28 to 30 degrees Celsius. The next day, inoculate 500 milliliters of plain LB media in a shaker flask with nine milliliters of overnight culture.Place the cultures on a shaker at 28 to 30 degrees Celsius. Following incubation and dividing the cultures into tubes, centrifuge the chilled tubes at four degrees Celsius and 4, 000 G for 15 minutes. Using a pipette, remove the supernatant and resuspend each pellet in 50 milliliters of ice cold water.After centrifuging and discarding the supernatant, resuspend the pellet in 25 milliliters of ice cold water in each tube. Centrifuge as before, then resuspend the pellet in one milliliter of ice cold 10%glycerol. Now, combine the contents of all the tubes into a single 50-milliliter tube before centrifuging.Finally, after discarding the supernatant, resuspend the pellet in 400 microliters of ice cold glycerol.

Research

JoVE Journal

Bioengineering

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...