electroporation of agrobacterium tumefaciens for genetic engineering of microalgal cells

利用根癌农杆菌在小球藻中的稳定基因整合

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.

作者聚焦：使用根癌农杆菌优化寻常小球藻的转化方案 

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

Watch this Scientific Journal Video about Electroporation of  Agrobacterium tumefaciens  for Genetic Engineering of Microalgal Cells at JoVE.com

Electroporation of  Agrobacterium tumefaciens  for Genetic Engineering of Microalgal Cells  

根癌农杆菌的电穿孔用于微藻细胞的基因工程

首先将 50 微升电感受态根癌农杆菌细胞加入预冷的 0.1 毫米 Q-vet 中。向 Q-vet 中加入 2 微升 pCAMBIA1302。现在使用 2， 400 伏的电穿孔仪对细胞进行电穿孔。立即向 Q-vet 中加入 1 毫升 LB 培养基，然后上下移液以轻轻混合。接下来，将重悬的细胞转移到 1.5 毫升微管中。然后在 24 至 30 摄氏度下孵育至少一小时以恢复。现在将细胞铺在补充抗生素的LB螺旋钻上。将培养板在 28 至 30 摄氏度下孵育 2 至 3 天。最后，在LB肉汤中接种单个菌落。让文化在一夜之间成长。然后用 50% 甘油溶液在 80 摄氏度下冷冻保存含质粒的菌株以备将来使用。

electroporation-agrobacterium-tumefaciens-for-genetic-engineering

Research

JoVE Journal

Bioengineering

Electroporation of Agrobacterium tumefaciens for Genetic Engineering of Microalgal Cells

175 次观看. 首先将 50 微升电感受态根癌农杆菌细胞加入预冷的 0.1 毫米 Q-vet 中。向 Q-vet 中加入 2 微升 pCAMBIA1302。现在使用 2， 400 伏的电穿孔仪对细胞进行电穿孔。立即向 Q-vet 中加入 1 毫升 LB 培养基，然后上下移液以轻轻混合。接下来，将重悬的细胞转移到 1.5 毫升微管中。然后在 24 至 30 摄氏度下孵育至少一小时以恢复。现在将细胞铺在补充抗生素的LB螺旋钻上。将培养板?...

视频: Electroporation of  Agrobacterium tumefaciens  for Genetic Engineering of Microalgal Cells  

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 the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

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