preparation of electrocompetent agrobacterium tumefaciens culture for microalgal genetic engineering

Chlorella vulgaris'te Agrobacterium tumefaciens Kullanılarak Kararlı Gen Entegrasyonu

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.

Yazar Spotlight: Chlorella vulgaris için Agrobacterium tumefaciens Kullanılarak Optimize Edilmiş Dönüşüm Protokolü 

Yeşil Mikroalglerin Agrobacterium tumefaciens Aracılı Genetik Mühendisliği ( Chlorella 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  

Mikroalgal Genetik Mühendisliği için Elektroyetkin Agrobacterium tumefaciens Kültürünün Hazırlanması

Agrobacterium tumefaciens kültürünün gece boyunca kültürlerinin 0,5 mikrolitresini 50 mililitre otoklavlanmış ultra saf su ile seyrelterek başlayın. Bu seyreltilmiş kültürün 10 mikrolitresini bir lizojen et suyu veya LB plakasına yayın. Plakayı bir ila üç gün boyunca 28 ila 30 santigrat derecede inkübe edin.Şimdi, tek bir koloniyi rifampisin ile desteklenmiş 20 mililitre LB besiyerinde aşılayın. Kültürü gece boyunca 28 ila 30 santigrat derecede inkübe edin. Ertesi gün, 500 mililitre düz LB ortamını dokuz mililitre gece kültürü ile bir çalkalayıcı şişesine aşılayın.Kültürleri 28 ila 30 santigrat derecede bir çalkalayıcıya yerleştirin. İnkübasyonun ve kültürlerin tüplere bölünmesinin ardından, soğutulmuş tüpleri dört santigrat derecede ve 4.000 G'de 15 dakika santrifüjleyin. Bir pipet kullanarak, süpernatanı çıkarın ve her peleti 50 mililitre buz gibi soğuk suda yeniden süspanse edin.Süpernatanı santrifüjledikten ve attıktan sonra, peleti her tüpte 25 mililitre buz gibi soğuk suda yeniden süspanse edin. Daha önce olduğu gibi santrifüjleyin, ardından peleti bir mililitre buz soğuk% 10 gliserol içinde yeniden süspanse edin. Şimdi, santrifüjlemeden önce tüm tüplerin içeriğini 50 mililitrelik tek bir tüpte birleştirin.Son olarak, süpernatanı attıktan sonra, peleti 400 mikrolitre buz gibi soğuk gliserol içinde yeniden süspanse edin.

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Research

JoVE Journal

Bioengineering

Preparation of Electrocompetent Agrobacterium tumefaciens Culture for Microalgal Genetic Engineering

189 Görüntüleme. Agrobacterium tumefaciens kültürünün gece boyunca kültürlerinin 0,5 mikrolitresini 50 mililitre otoklavlanmış ultra saf su ile seyrelterek başlayın. Bu seyreltilmiş kültürün 10 mikrolitresini bir lizojen et suyu veya LB plakasına yayın. Plakayı bir ila üç gün boyunca 28 ila 30 santigrat derecede inkübe edin.Şimdi, tek bir koloniyi rifampisin ile desteklenmiş 20 mililitre LB besiyerinde aşılayın. Kültürü gece boyunca 28 ila 30 santigrat derecede inkübe edin...

Video: 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 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 ...