preparation of electrocompetent agrobacterium tumefaciens culture for microalgal genetic engineering

Stabiele genintegratie in Chlorella vulgaris met behulp van 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.

Auteur in de schijnwerpers: geoptimaliseerd transformatieprotocol voor Chlorella vulgaris met behulp van Agrobacterium tumefaciens 

Agrobacterium tumefaciens-gemedieerde genetische manipulatie van groene microalgen, 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 Preparation of Electrocompetent  Agrobacterium tumefaciens  Culture for Microalgal Genetic Engineering at JoVE.com

Preparation of Electrocompetent  Agrobacterium tumefaciens  Culture for Microalgal Genetic Engineering  

Bereiding van elektrocompetente Agrobacterium tumefaciens-cultuur voor genetische manipulatie van microalgen

Begin met het verdunnen van 0,5 microliter van de nachtculturen van de Agrobacterium tumefaciens-cultuur met 50 milliliter geautoclaveerd ultrapuur water. Verdeel 10 microliter van deze verdunde cultuur over een lysogene bouillon of LB-plaat. Incubeer de plaat één tot drie dagen bij 28 tot 30 graden Celsius.Inoculeer nu een enkele kolonie in 20 milliliter LB-media aangevuld met rifampicine. Incubeer de cultuur 's nachts bij 28 tot 30 graden Celsius. Inoculeer de volgende dag 500 milliliter gewone LB-media in een schudkolf met negen milliliter nachtcultuur.Plaats de culturen op een shaker bij 28 tot 30 graden Celsius. Na de incubatie en de verdeling van de culturen in buisjes, centrifugeert u de gekoelde buisjes gedurende 15 minuten bij vier graden Celsius en 4.000 G. Verwijder met een pipet het supernatans en resuspendeer elke pellet in 50 milliliter ijskoud water.Na het centrifugeren en weggooien van het supernatant, resuspendeert u de pellet in 25 milliliter ijskoud water in elke buis. Centrifugeer zoals voorheen en resuspendeer de pellet vervolgens in een milliliter ijskoude 10% glycerol. Combineer nu de inhoud van alle buisjes in een enkele buis van 50 milliliter voordat u gaat centrifugeren.Eindelijk, na het weggooien van het supernatant, resuspendeert u de pellet in 400 microliter ijskoude glycerol.

Research

JoVE Journal

Bioengineering

Preparation of Electrocompetent Agrobacterium tumefaciens Culture for Microalgal Genetic Engineering

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

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

Electroporation of Agrobacterium tumefaciens for Genetic Engineering of Microalgal Cells

Electroporation of  Agrobacterium tumefaciens  for Genetic Engineering of Microalgal Cells  

Elektroporatie van Agrobacterium tumefaciens voor genetische manipulatie van microalgencellen

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)  

Agrobacterium-gemedieerde transformatie van Chlorella vulgaris en bevestiging van genintegratie via kolonie-PCR (cPCR)