Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae
Abstract
:1. Introduction
2. Results and Discussion
2.1. Construction and Transformation Efficiency of Several Multicistronic APHVIII-based Fusion Plasmids
2.2. Phyco69 Enables the Selection of Transformants with High Expression Levels of the Gene of Interest
2.3. Phyco69 Enables the Simultaneous Expression of Two Genes, Which are Efficiently Processed Generating Independent Proteins
3. Materials and Methods
3.1. Strains and Culture Conditions
3.2. Plasmids Constructions
3.3. Chlamydomonas Nuclear Transformation
3.4. Real Time PCR
3.5. Western Blot
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
Target Gene | Primer Name | Sequence |
---|---|---|
BLE | qBLE-F | CGACTTCGCCGGTGTGGTC |
qBLE-R | CACGAAGTGCACGCAGTTGC | |
APHVIII | qAPHVIIIF | GAGGATCTGGACGAGGAGCGGAA |
qAPHVIIIR | CCCTCAGAAGAACTCGTCCAACAGC | |
UBC8 | qUBC8 | GTACAGCGGCGGCTAGAGGCAC |
qUBC8 | AGCGTCAGCGGCGGTTGCAGGTATCT |
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Plasmid | 200 ng | 500 ng | 1 µg | 2 µg | Transformants µg−1 DNA |
---|---|---|---|---|---|
Phyco69 | 98 ± 22 | 215 ± 38 | 380 ± 58 | 820 ± 70 | 427 |
pSI103 | 28 ± 12 | 60 ± 21 | 140 ± 32 | 250 ± 45 | 131 |
PhycoC67 | 0 | 5 ± 2 | 7 ± 4 | 18 ± 6 | 6.5 |
PhycoC67FL | 0 | 8 ± 4 | 12 ± 7 | 3.5 |
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Molina-Márquez, A.; Vila, M.; Rengel, R.; Fernández, E.; García-Maroto, F.; Vigara, J.; León, R. Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae. Int. J. Mol. Sci. 2020, 21, 718. https://doi.org/10.3390/ijms21030718
Molina-Márquez A, Vila M, Rengel R, Fernández E, García-Maroto F, Vigara J, León R. Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae. International Journal of Molecular Sciences. 2020; 21(3):718. https://doi.org/10.3390/ijms21030718
Chicago/Turabian StyleMolina-Márquez, Ana, Marta Vila, Rocío Rengel, Emilio Fernández, Federico García-Maroto, Javier Vigara, and Rosa León. 2020. "Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae" International Journal of Molecular Sciences 21, no. 3: 718. https://doi.org/10.3390/ijms21030718