Team:NYMU-Taipei/Demonstrate
Demonstration
Team NYMU Taipei this year accentuates on the enhancement of oil accumulation in microalgae through genetic manipulation and stress condition cultivation. Several achievements and criteria have been attained during this long odyssey. The followings are brief results of our project:
(1) A well-documented microalgae transformation platform has been created
We have constructed a backbone, pPIGBACK, particularly compatible for microalgae to transform foreign genes into the cell by triggering gene double-crossover homologous recombination. CRTZ gene has been successfully transformed into microalgae and sparked a cascade of chemical reaction to produce Zeaxanthin. Moreover, the transformation accuracy is 52%, which clearly points out the roaring success of the construction. The appearance of the transformants are significantly different from that of the wild-type.
See more detail: Pigments - Conclusion
(2) High-affinity protein NrtA system combined with suicide mechanism has been successfully implemented
NrtA gene is extracted from Synechocystis sp. PCC6803 and transformed into E. coli for mass production of the high-affinity protein. Combined with semi-permeable and protein extraction system, the amount of nitrate and nitrite in the microalgae medium has declined, which has firmly proved our concept. The concept is further applicable to our nitrogen starvation plan.
Taking biosafety under consideration, an inducible suicide mechanism based on the combination of endolysin and holing has been constructed and undergone a functional test. The suicide mechanism has been proved valid by a cooperative functional test conducted by team TAS Taipei and us.
See more detail: Nitrogen Starvation - Suicide Mechanism Functional Test
(3) Insights gained from modeling have been effectively applied to our experiments
Several modeling, simulation and data analysis have been conducted along with wet lab experiments. Results of modeling have been considered when determining conditions of experiments. As far as our project is concerned, it is not exaggerated to say that successfully culturing our microalgae is prior to all other experiments. Precise growth curve should be illustrated before any experiment related to microalgae was carried out. Conspicuously, the simulated growth curve is highly correlated to the experimental one.
(4) A myriad of pigment sequences has been investigated and constructed
Photosynthetic efficiency is crucial to growth and oil accumulation in microalgae. In our project, several aspects including synthesis, expression and characteristics were deeply investigated. Genes related to the pigments have been constructed. We intended to transform pigment sequences extracted from other species into microalgae to compare wavelength absorbance and photosynthetic efficiency between color combination.
This figure is the variation of starch content per cell per day of CrtZ transformants. The result implied that CrtZ transformants could produce more starch per day than the wild type and had better photosynthetic efficiency.
See more detail: Pigments
(5) NrtA protein has been proved valid
Photosynthetic efficiency is crucial to growth and oil accumulation in microalgae. In our project, several aspects including synthesis, expression and characteristics were deeply investigated. Genes related to the pigments have been constructed. We intended to transform pigment sequences extracted from other species into microalgae to compare wavelength absorbance and photosynthetic efficiency between color combination.
The bar figure shows nitrate concentration of cell lysate, and the table represents Dunnett’s T3 test of nitrate concentration of cell lysate. Both of the result clearly demonstrate that nitrate and nitrite concentration of competent cell and NrtA was significant different, which verifies NrtA protein’s capability of capturing nitrite and nitrate.
See more detail: Nitrogen Starvation - NrtA Functional Test
(6) Pigmentation genes collection
Aside from CrtZ gene, we also obtained other pigmentation genes from distribution kits and bacterium. PCR products were successfully acquired through templates and well-designed primers. The gel electrophoresis results demonstrate our effort to construct the pigmentation genes collection. The followings are MelA, Lycopene’s upstream, Lycopene’s downstream and IndC gene.
See more detail: Pigments
(7) Microalgae gene amplification
To confirm the correctness of CrtZ transformation, we extracted the genomic DNA of Synechococcus elongatus PCC 7942 as the template for PCR Test. The genomic DNA was successfully extracted with liquid nitrogen and tissue grinder. Further PCR test was performed and the result was shown below. The transformation accuracy is up to 52%, which was a crucial achievement of our project.
