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Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris
In This Article
Summary
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.
Abstract
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.
Introduction
Agrobacterium tumefaciens, a gram-negative soil-borne bacterium, possesses a unique interkingdom gene transfer ability, earning it the title "natural genetic engineer"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. Ho....
Protocol
All media and solutions must be autoclaved prior to use unless otherwise stated. All centrifuge tubes, pipette tips, etc., should be sterile or autoclaved before use. For easy reference, the media recipes used in this protocol are listed in Table 1.
1. Preparation of A. tumefaciens electrocompetent cells
- Inoculate Agrobacterium (AGL-1) into a 25 mL sterile shaker flask of LB media (supplemented with rifampicin, 20 mg/L-1
Representative Results
To show successful transformation using the method above, C. vulgaris was cocultured with either AGL-1 containing the pCAMBIA1302 plasmid or without the plasmid (wild-type and plated on TAP agar supplemented with Hygromycin B and cefotaxime (Figure 1A). The leftmost plate shows the transformed colonies capable of growth on Hygromycin B/cefotaxime plates, and the middle plate shows that wild-type AGL-1 cannot grow on the Hygromycin B/cefotaxime plates. The rightmost plate shows that .......
Discussion
The efficiency of transformation is associated with several different parameters. The choice of A. tumefaciens strains used for AMT is crucial. AGL-1 is one of the most invasive strains discovered and, for this reason, has been routinely used in plant AMT. Supplementing the induction media with glucose (15-20 mM) is also important for AMT efficiency. Considering C. vulgaris can grow in both phototrophic and heterotrophic conditions, glucose or other carbon sources are often omitted from microalgae media.......
Acknowledgements
The authors would like to thank Prof. Paul Hooykaas for kindly providing the pCAMBIA1302 vector and Agrobacterium tumefaciens AGL1 from the Institute of Biology Leiden, Leiden University, the Netherlands. The authors would also like to thank Eva Colic for her help in growing the fluorescent transformants. This work was funded by the Natural Sciences and Engineering Research Council of Canada and the Mitacs Accelerate program.
....Materials
| Name | Company | Catalog Number | Comments |
| 1 Kb Plus DNA ladder | FroggaBio | DM015 | |
| Acetosyringone | Fisher Scientific | D26665G | |
| Agrobacterium tumefaciens | Gold Biotechnologies | Strain: AGL-1; Gift from Prof. Paul Hooykaas | Genotype: C58 RecA (RifR/CarbR) pTiBo542DT-DNA |
| Biotin | Enzo Life Sciences | 89151-400 | |
| CaCl2·2H2O | VWR | BDH9224-1KG | |
| Cefotaxime | AK Scientific | J90010 | |
| Chlorella vulgaris | University of Texas at Austin Culture Collection of Algae | Strain: UTEX 395 | Wildtype strain |
| CoCl2·6H2O | Sigma Aldrich | C8661-25G | |
| CuSO4·5H2O | EMD Millipore | CX2185-1 | |
| FeCl3·6H2O | VWR | BDH9234-500G | |
| Gene Pulser Xcell Electroporator | Bio-Rad | 1652662 | Main unit equipped with PC module. |
| GeneJET Plant Genome Purification Kit | Thermo Scientific | K0791 | |
| Glacial acetic acid | VWR | CABDH3093-2.2P | |
| Glycerol | BioBasic | GB0232 | |
| HEPES Buffer | Sigma Aldrich | H-3375 | |
| Hygromycin B | Fisher Scientific | AAJ6068103 | |
| K2HPO4 | VWR | BDH9266-500G | |
| Kanamycin | Gold Biotechnologies | K-250-25 | |
| KH2PO4 | VWR | BDH9268-500G | |
| MgSO4·7H2O | VWR | 97062-134 | |
| MnCl2·4H2O | JT Baker | BAKR2540-01 | |
| Na2CO3 | VWR | BDH7971-1 | |
| Na2EDTA·2H2O | JT Baker | 8993-01 | |
| Na2MoO4·2H2O | JT Baker | BAKR3764-01 | |
| NaCl | VWR | BDH7257-7 | |
| NaH2PO4 H2O | Millipore Sigma | CA80058-650 | |
| NaNO3Â | VWR | BDH4574-500G | |
| NEBExpress Ni Resin | NewEngland BioLabs | NEB #S1427 | |
| NH4Cl | VWR | BDH9208-500G | |
| pCAMBIA1302 | Leiden University | Gift from Prof. Paul Hooykaas | pBR322, KanR, pVS1, T-DNA(CaMV 35S/HygR/CaMV polyA, CaMV 35S promoter/mgpf5-6xhis/NOS terminator) |
| Polypropylene Columns (5 mL) | QIAGEN | 34964 | |
| Precision Plus Protein Unstained Protein Standards, Strep-tagged recombinant, 1 mL | Bio-Rad | 1610363 | |
| Rifampicin | Millipore Sigma | R3501-1G | |
| SunBlaster LED Strip Light 48 Inch | SunBlaster | 210000000906 | |
| Synergy 4 Microplate UV/Vis spectrometer | BioTEK | S4MLFPTA | |
| Tetracycline | Thermo Scientific Chemicals | CAAAJ61714-14 | |
| TGX Stain-Free FastCast Acrylamide Kit, 12% | Bio-Rad | 1610185 | |
| Thiamine | TCI America | T0181-100G | |
| Tris Base | Fisher Scientific | BP152-500 | |
| Tryptone | BioBasic | TG217(G211) | |
| Vitamin B12 (cyanocobalamin) | Enzo Life Sciences | 89151-436 | |
| Yeast Extract | BioBasic | G0961 | |
| ZnSO4·7H2O | JT Baker | 4382-01 |
References
- Smith, E. F., Townsend, C. O. A plant tumor of bacterial origin. Science. 25 (643), 671-673 (1907).
- Chilton, M. D., et al. Stable incorporation of plasmid DNA into higher plant cells: The molecular basis of tumorigenesis.....
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