electroporation of agrobacterium tumefaciens for genetic engineering of microalgal cells

שילוב גנים יציב בכלורלה וולגריס באמצעות 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: פרוטוקול טרנספורמציה ממוטב עבור Chlorella vulgaris באמצעות Agrobacterium tumefaciens 

Agrobacterium tumefaciens-מתווך גנטי של מיקרו-אצות ירוקות, 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 Electroporation of  Agrobacterium tumefaciens  for Genetic Engineering of Microalgal Cells at JoVE.com

Electroporation of  Agrobacterium tumefaciens  for Genetic Engineering of Microalgal Cells  

אלקטרופורציה של גידולי אגרובקטריום להנדסה גנטית של תאי מיקרו-אצות

התחל על ידי הוספת 50 מיקרוליטר של תאים Agrobacterium tumefaciens electrocompetent לתוך Q-vet 0.1 מ"מ מקורר מראש. הוסף שני מיקרוליטרים של pCAMBIA1302 לווטרינר Q. עכשיו להשתמש אלקטרופורטור ב 2, 400 וולט כדי לחשמל את התאים.הוסיפו מיד מיליליטר אחד של מדיית LB לווטרינר Q-vet ופיפטה את התמיסה למעלה ולמטה כדי לערבב בעדינות. לאחר מכן, להעביר את התאים resuspended לתוך microtube 1.5 מיליליטר. לאחר מכן לדגור אותו ב 24 עד 30 מעלות צלזיוס במשך מינימום של שעה אחת כדי להתאושש.עכשיו צלחת את התאים על אנטיביוטיקה בתוספת LB auger. דוגרים על הצלחות בטמפרטורה של 28 עד 30 מעלות צלזיוס למשך יומיים-שלושה. לבסוף, לחסן מושבה אחת במרק LB.אפשרו לתרבות לצמוח בן לילה. לאחר מכן שמרו בהקפאה את הזן המכיל פלסמיד עם תמיסת גליצרול 50% בטמפרטורה של 80 מעלות צלזיוס לשימוש עתידי.

Research

JoVE Journal

Bioengineering

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 ...

Preparation of Electrocompetent Agrobacterium tumefaciens Culture for Microalgal Genetic Engineering

Preparation of Electrocompetent  Agrobacterium tumefaciens  Culture for Microalgal Genetic Engineering  

הכנת תרבית גידולי אגרובקטריום אלקטרו-מוכשרים להנדסה גנטית של מיקרו-אצות

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)  

טרנספורמציה בתיווך אגרובקטריום של כלורלה וולגריס ואישור אינטגרציה גנטית באמצעות PCR מושבה (cPCR)