agrobacterium-mediated transformation of chlorella vulgaris and confirmation of gene integration via colony pcr (cpcr)

Stable Gene Integration in &lt;em&gt;Chlorella vulgaris&lt;/em&gt; Using &lt;em&gt;Agrobacterium tumefaciens&lt;/em&gt;

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.

Watch this Scientific Journal Video about Agrobacterium-Mediated Transformation of Chlorella vulgaris  and Confirmation of Gene Integration via Colony PCR CPCR at JoVE.com

Agrobacterium-Mediated Transformation of Chlorella vulgaris  and Confirmation of Gene Integration via Colony PCR (CPCR)  

Agrobacterium-Mediated Transformation of Chlorella vulgaris and Confirmation of Gene Integration via Colony PCR (cPCR)

Begin by pipetting out 0.5 milliliters of a log phase culture of chlorella vulgaris. Spread the culture on at Tris-Acetate-Phosphate or TAP Agar plate. Grow the culture for five days at 25 degrees Celsius.Next, inoculate 10 milliliters of supplemented LB broth with a loop full of electroporated agrobacterium tumefaciens culture in a shaker flask. Incubate the flask at 28 to 30 degrees Celsius and 250 RPM overnight. The next day, inoculate one milliliter of the overnight culture into 50 milliliters of supplemented LB.Incubate the flask at 28 to 30 degrees Celsius and 250 RPM.To co-cultivate the algal and bacterial cultures. First, transfer the agrobacterium culture into a 50 milliliter tube. Centrifuge the tube at 4, 000 G for 30 minutes at room temperature.Then, pipette out the supernatant to discard it and wash the cells twice using the induction medium. Next, add 25 milliliters of induction media onto the Chlorella Vulgaris culture plate. Transfer the cells into a 50 milliliter tube, then centrifuge at 4, 000 G for 15 minutes at room temperature.After disposing of the supernatant, combine the algal cell pellet with 200 microliters of the bacterial suspension. Shake the combined culture in a rotary incubator at 21 to 25 degrees Celsius, said at 150 RPM for one hour. Spread 200 microliters of the mixed culture onto induction medium plates, supplemented with 15 millimoles of glucose and incubate the plates in the dark at 21 to 25 degrees Celsius for three days.After three days, collect the microalgae into a flask using 10 milliliters of TAP media. Supplemented with 20 milligrams per liter of tetracycline. Incubate the flask in the dark for two days, maintaining a temperature between 21 to 25 degrees Celsius.Plate 500 microliters of the culture onto selective media supplemented with 20 milligrams per liter of tetracycline. Incubate the plates at 21 to 25 degrees Celsius in darkness for two days, before shifting them into an illuminated chamber. Select single colonies from the transformation plate and streak them onto TAP agar plates.To perform colony PCR, start by adding a small volume of the transformant algal cells to 10 microliters of sterile water. Boil the solution at 98 degrees Celsius for 15 minutes. Similarly process another PCR template for the confirmation of agrobacterium absence in the sample.Run the PCR samples on a DNA agarose gel with a ladder, to verify the size of the resulting fragments. Transformed colonies were able to grow on plates containing hygromycin with cefotaxime. The wild type colonies did not grow on the plates.Colonies resistant up to 70 milligrams per liter of cefotaxime were obtained. Colony PCR Amplicon of pCAMBIA1302 was absent in the algal samples. However MGFP5G in amplicon was detected in all three algal samples.Algal cultures that were repeatedly subcultures of Cefotaxime showed the absence of the virulence protein E2, indicative of plasmid absence. A significant difference in algal growth of the transformants and wild type strains were observed. Despite lower growth, the transformant had higher fluorescence levels when normalized for cell density.

agrobacterium-mediated-transformation-chlorella-vulgaris-confirmation

Research

JoVE Journal

Bioengineering

223 Views.  University of Waterloo. Begin by pipetting out 0.5 milliliters of a log phase culture of chlorella vulgaris. Spread the culture on at Tris-Acetate-Phosphate or TAP Agar plate. Grow the culture for five days at 25 degrees Celsius.Next, inoculate 10 milliliters of supplemented LB broth with a loop full of electroporated agrobacterium tumefaciens culture in a shaker flask. Incubate the flask at 28 to 30 degrees Celsius and 250 RPM overnight. The next day, inoculate one milliliter of the overnight culture into 50...