Team:SCUT-ChinaA/Results
Construction
We have successfully constructed a series of gene expression modules applying various synthetic biology techniques and successfully integrated them into our chassis. The correct integration and transcription of modules were verified by means of colony PCR, yeast genome extraction verification and quantitative PCR. Finally, we got four successful transformants that could be used for our subsequent fermentation.
Figure 1: the linear amplification plot of Po1f/lim-tESS. A) the result of delta Rn vs Cycle of NDPS1 in 1:1, B) the result of delta Rn vs Cycle of arLS in 1:1
Fermentation
After the successful transformants were obtained, they were inoculated into the YPD medium for two-phase fermentation. During the fermentation, we took samples every day for six days, and we got the growth curve of the strains, which LS gene from different species were introduced into, respectively. Since the growth of the strains is not much different, we finally selected the arLS gene (from Agastache rugosa) which limonene yields higher, reported in the literature, instead of the msLS gene (from Mentha spicata).
Figure 2: The growth curve of engineered strains in different medium. YPD represents the growth curve of the strain growing in medium using glucose as a carbon source. YPG represents the medium using glycerol as a carbon source. The arLS represents the strain which LS gene from Agastache rugosa is introduced into. The msLS represents the strain which LS gene from Mentha spicata is introduced into.
Detection
After fermentation, the fermentation samples were centrifuged to extract n-dodecane layer. Dodecane extracts our product limonene from the fermentation water phase. The limonene content in organic phase can be detected by gas chromatography. We measured the peak area and retention time of a series of different concentrations of limonene and drew a standard curve. The output of the product can be obtained by comparing the peak area of the product with the standard curve.
Figure 2: The growth curve of engineered strains in different medium. YPD represents the growth curve of the strain growing in medium using glucose as a carbon source. YPG represents the medium using glycerol as a carbon source. The arLS represents the strain which LS gene from Agastache rugosa is introduced into. The msLS represents the strain which LS gene from Mentha spicata is introduced into.
But after several times of fermentation and detection, we still failed to detect the limonene. One of the reasons we analyzed was that exogenous gene expression was inhibited, thereby product yield was not as expected. As so low the yield of the product is, the analysis method we currently use cannot detect the limonene. There is another possibility. The biological chassis we use is not well studied. In the process of our experiment, we found that many modules whose integration and transcription were verified successfully, were lost so fast that all the re-verification failed after fermentation. We are still working on this problem.
Discussion
During the implementation of our entire project, we verified the correct integration of modules by means of colony PCR, yeast genome extraction and quantitative PCR. For yeast strains, we determined the growth curve of the strain after the integration of the expression cassettes. In the aspect of limonene detection, we plotted the standard curve of limonene to prepare for the quantitative detection of limonene. During our experiments, we found that our expression modules integrated into the yeast genome were not as stable as we expected. After culturing in solid medium or liquid medium for a period of time, the engineered strains are easily degraded and our cassettes are also lost. Therefore, at the stage of gas chromatography detection, it is difficult to detect the product. We also need to do more research on Yarrowia lipolytica to better break through the difficulties we face today.
