In this year's project, 2020 SCU-China designed two sets of polycistronic systems in eukaryotes in order to produce ABA with Saccharomyces cerevisiae in high efficiency. We compared the differences between the 2A system and CRISPR-Csy4 system through fluorescence observation, protein expression and RT-qPCR, and finally selected the 2A system as the expression platform of BcABA genes for now. With the help of this system, we successfully expressed four BcABA family proteins using single plasmid and detected ABA production by HPLC.
1. Functional verification of the expression of polycistrons in 2A system
In order to ensure that 2A system can effectively perform its polycistronic expression function in expressing BcABA gene family when transformed into Saccharomyces cerevisiae, we added NLS sequence before yeGFP and T2A sequence between yeGFP and DsRed. According to our design, if the T2A sequence can separate NLS-yeGFP from DsRed, we should be able to observe green nuclei and red cytoplasm under inverted fluorescence microscope.
As shown in figure 1, after transform the plasmid pY26TEF-GDP with NLS-yeGFP-T2A-DsRed(BBa_K3544206 & BBa_K3544504) into Saccharomyces cerevisiae and express the proteins, we can observe that NLS-yeGFP transports into the nucleus clearly under 488 nm excitation light, while the entire cell with red fluorescence under the 560 nm excitation light (shooting environment is set to 1000 x magnification, 100 ms, 64 times the brightness gain).
Fig 1. The results were observed through excitation light of 488nm and 560nm in bright field under inverted fluorescence microscope. From the top to the bottom: EGY48 which had not been transferred to plasmids, EGY48 which had been transferred to pTEF1-NLS-yeGFP-T2A-DsRed-tCYC1 (BBa_K3544504), and EGY48 which had been transferred to pGAP-NLS-yeGFP-T2A-DsRed-tCYC1 (BBa_K3544206)
But at the same time, we also found that DsRed, which should not enter the nucleus, also did that, and even showed the phenomenon of enrichment. Based on the description of 2A sequence in the literature, we speculate that this is caused by the incomplete cutting efficiency of 2A sequence. To verify this hypothesis, we extracted total protein from Saccharomyces cerevisiae and carried out WesternBlot experiments using anti-GFP Rabbit Polyclonal Antibody.
Fig 2. WesternBlot results: from left to right: EGY48 without plasmid, EGY48 which had been transferred with pTEF1-NLS-yeGFP-T2A-DsRed-tCYC1 (BBa_K3544504), and EGY48 which had been transferred with pGAP-NLS-yeGFP-T2A-DsRed-tCYC1 (BBa_K3544206)
The results of WesternBlot showed that two obvious bands were observed at 57kDa (NLS-yeGFP-T2A-DsRed) and 37kDa (NLS-yeGFP-T2A). These results confirm our hypothesis that the fluorescence of DsRed in the nucleus is indeed caused by fusion proteins. In order to verify its cleavage efficiency, Image J was used to process the image. Finally, it was found that the cleavage efficiency of T2A under pTEF1 and pGAP was about 58% and 51%, respectively (view Measurement for details).
2. Function verification of CRISPR/Csy4 system polycistron expression
In order to verify whether our CRISPR-Csy4 system could successfully cleave mRNA and express protein in Saccharomyces cerevisiae, we assembled pTEF1-NLS-Csy4-tADH1 / pGAP-NLS-yeGFP-CRISPR-DsRed-tCYC1 (BBa_K3544503 & BBa_K3544888) on pY26TEF-GDP. As an intermediate plasmid, pGAP-CRISPR-DsRed-tCYC1 (BBa_K3544204) was also transformed to EGY48, which can be used to simulate the expression of DsRed in Saccharomyces cerevisiae. On the other hand, it can be used to prove that the nuclear entrance of DsRed in 2A system is indeed caused by fusion protein.
Fig 3. The results were observed through excitation light of 488nm and 560nm in bright field under inverted fluorescence microscope. From the top to the bottom: pTEF1-NLS-Csy4-tADH1 / pGAP-NLS-yeGFP-CRISPR-DsRed-tCYC1 (BBa_K3544503 & BBa_K3544888) in EGY48 and pGAP-CRISPR-DsRed-tCYC1 (BBa_K3544204) in EGY48
As shown in the figure above (Fig 3), CRISPR-Csy4 can express the NLS-yeGFP protein which successfully enter the nucleus, while CRISPR-DsRed is uniformly distributed in the cell without entering the nucleus (the shooting environment was set at 1000x magnification, 100ms, and 64-times brightness gain). In order to compare the protein expression levels of CRISPR-Csy4 system and 2A system, we conducted the following WesternBlot experiment.
Fig 4. WesternBlot Results：From left to right:EGY48 without plasmid，pTEF1-NLS-yeGFP-T2A-DsRed-tADH1（BBa_K3544504）transformed in EGY48，pGAP-NLS-yeGFP-T2A-DsRed-tCYC1（BBa_K3544206）transformed in EGY48，pGAP-CRISPR-DsRed-tCYC1 (BBa_K3544204) transformed in EGY48, and pTEF1-NLS-Csy4-tADH1 / pGAP-NLS-yeGFP-CRISPR-DsRed-tCYC1 (BBa_K3544503 & BBa_K3544888) transformed in EGY48
After Image J processed, it was found that the relative protein content at 28kDa of lane in CRISPR-Csy4 system was much higher than the sum of NLS-yeGFP-T2A-DsRed and NLS-yeGFP-T2A in 2A system (view Measurement for details). The results showed the advantage of CRISPR-Csy4 system in protein expression.
3. Csy4 cutting efficiency verification
The cleavage efficiency of Csy4 to mRNA is still unknown in S. cerevisiae. Besides, the function of Csy4 needs to be confirmed in our project to verify that our design is available. We successfully constructed the plasmids of Csy4 and inserted its CRISPR hairpin between yeGFP and DsRed. Then, we verified the transcriptional level of fluoresce proteins via RT-qPCR and calculate the cleavage efficiency of Csy4 (view Measurement for details).
Fig 5. The result of RT-qPCR
4. ABA production using the BcABA protein family in Saccharomyces cerevisiae
To express four BcABA proteins from Botrytis cinerea using a single plasmid in S. cerevisiae, we divided the four BcABA genes into two groups (view ABA Model for details) and assembled pTEF1-BcABA3-T2A-BcABA2-tADH1 (BBa_K3544777) and pGAP-BcABA1-T2A-BcABA4-tCYC1 (BBa_K3544666) into pY26TEF-GDP. Initially, we wanted to use double enzyme digestion. However, after completing the assembly of BcABA3-T2A-BcABA2, we attempted to assemble BcABA1-T2A-BcABA4 into the plasmid repeatedly but failed.
We thought this may due to the low efficiency of T4 ligation in long fragment—the linear pY26TEF-GPD with BcABA3/2 is about 10kb. After failures, we adjusted our strategy from double enzyme digestion-ligation to Gibson assembly and finally assembled BcABA1-T2A-BcABA4 into the ORF of pGAP(Figure 6).
Fig 6.The result of enzyme digestion
M:Takara DL5000 Marker 1:pY26TEF-GDP
2:enzyme digested by NotI,BglII,HindIII,BamHI
3:enzyme digested by NotI,BglII
4:enzyme digested by HindIII,BamHI
Finally, after 5 days of incubation at 29℃, ABA concentration in fermentation broth was measured by HPLC. The peak area was calculated to obtain the ABA concentration of 141±20 μg/L in the fermentation broth with 110mg/L ABA as the control (view Measurement for details).
Fig 7. The result of HPLC
