Part:BBa_K3544015
Improved T2A
T2A is a kind of 2A sequence from Thosea asigna virus, which can relize the cleavage of multiple genes in eukaryotes. It has been proved to achieve bicistronic gene expression in S. cerevisiae by 2020 SCU-China. We add a pair of spacers at the front and back of T2A sequence for better cleavage effciency.
BBa_K3544106 was constructed on the pY26TEF-GDP, and the cleavage efficiency of T2A system (BBa_K3544504 & BBa_K3544206) in Saccharomyces cerevisiae was measured to be 57.98% under pGAP and 50.52% under pTEF1.
In this part, western blot (Fig 1) was used for the identification of protein expression and 2A cleavage, and then Image J was used for the quantitative calculation to analyze the expression level of the protein. In addition, we used GADPH as the reference gene to calculate the relative expression level.
Fig 1. Western Blot Result. (from left to right) lane1：NC，lane 2：pGAP-NLS-yeGFP-T2A-DsRed，lane 3：pTEF1-NLS-yeGFP-T2A-DsRed，lane 4：pTEF1-DsRed，lane5：pTEF1-NLS-yeGFP.
Firstly, background subtraction and color inversion are processed by Image J (Fig 8). Then we choose integrated optical density (IOD) as the measurement to represent the expression level, which can eliminate the influence of the selected area on the grayscale to a certain extent. However, we found that the difference in area still had some influence on the measuring value, so we used the same area for the selection of the strip. Since the target proteins of band 2 and 3 are the same, for the trailing strip, we merely measure the part which is at the same position of the right clear one.
Fig 2. Image processed by Image J
The original data of the determination is shown in the figure below (Fig 9). It is not difficult to find that the expression level of NLS-yeGFP (PTEF1-CRISPR) is much higher than that of the 2A system (including fusion protein and cleaved protein). Considering that the expression levels of the reference protein (GADPH) are not the same among the experimental groups, we decided to use relative expression levels (target protein IOD/reference protein IOD) to characterize the expression level for subsequent analysis.
Fig 3. The measurement of raw data
From the calculation results (Fig 9), we noticed that the total relative expression of pGAP-NLS-yeGFP-T2A-DsRed is 3.97, and the total relative expression of pTEF1-NLS-yeGFP-T2A-DsRed is 4.26, which is consistent with those measured by the enzyme-labeled instrument. The results also indicate that the strength of pTEF1 is slightly higher than pGAP. It is worth noting that the total relative expression of CRISPR-Csy4 system is much higher than that in 2A system (10.3 times).
Fig 4. Calculation of relative expression
Finally, we measured the cleavage efficiency of 2A sequence. Define the cleavage efficiency as the ratio of the relative expression of the cleaved protein to the total relative expression, we found that the cleavage efficiency of 2A sequence in pGAP-NLS-yeGFP-T2A-DsRed was 57.98%, and that of 2A sequence in pTEF1-NLS-yeGFP-T2A-DsRed was 50.52%.
To sum up, the above conclusions can be drawn from measurement in this part: 1. The strength of pTEF1 is higher than that of pGAP in S. cerevisiae. 2. The upstream protein expression level of polycistrons in CRISPR-csy4 system is much higher than that in 2A system. 3. The cleavage efficiency of T2A sequence in S. cerevisiae is 50%-60%.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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