1. Measure the promoter strength of pTEF1 and pGAP
BBa_K3544204 and BBa_K3544206 were constructed on the pY26TEF-GDP, and the promoter strength of pTEF1 and pGAP in Saccharomyces cerevisiae was estimated by the fluorescence intensity of yeGFP(excitation wavelength 485nm, emission wavelength 515nm) and DsRed(excitation wavelength 580nm, emission wavelength 615nm).
Step1. Choose 96-well plate: for fluorescence intensity detection, we should choose black plate to absorbs all the miscellaneous light that is refracted and reflected in every well, to lower the background.
Step2. Detect the OD of cells, or normalize the OD of every sample. In our study, we normalize the OD into 1.
Step3. Collect cells in one plate as a consistent form. For example, in our study, we divided 100μl in every well.
Step4.Detect fluorescence intensity.
Fig 1. Raw data of yeGFP fluorescence intensity
Fig 2. Raw data of DsRed fluorescence intensity
Step5. Calculate relative fluorescence intensity, analyze data. Choose a data of SD-Ura as standard data (for example, 2084 for yeGFP), and divide every data to 2084 when consider yeGFP:
Fig 3. Data processing of yeGFP fluorescence intensity
And DsRed:
Fig 4. Data processing of DsRed fluorescence intensity
Step6. Draw graph, and analyze data by Ordinary one-way ANOVA by GraphPad Prism 7.00.
Fig 5. Bar chart and ANOVA result of DsRed fluorescence intensity
Fig 6. Bar chart and ANOVA result of yeGFP fluorescence intensity
Conclusion: Promoter pTEF1 is stronger than pGAP.
2. NLS-yeGFP-2A-DsRed expression level and 'cleavage' efficiency of 2A sequence
BBa_K3544106 was constructed on the pY26TEF-GDP, and the cleavage efficiency of T2A system (BBa_K3544204 & BBa_K3544206) in Saccharomyces cerevisiae was estimated to be 57.98% under pGAP and 50.52% under pTEF1.
In this part, western blot (Fig 7) 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 7. 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 8. Image processed by Image J
$$IOD=lg(\frac{The\ average\ grayscale\ of\ the\ brightest\ region\ of\ the\ image}{The\ average\ grayscale\ of\ the\ target\ area\ to\ be\ measured})*Area$$
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 9. 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 10. 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%.
3. CRISPR-Csy4 cleavage efficiency
BBa_K3544105 was constructed on the pY26TEF-GDP, and the cleavage efficiency of CRISPR-Csy4 system (BBa_K3544888 & BBa_K3544503) in S. cerevisiae was estimated to be 98.17%.
The transcriptional level was measured by qPCR in a CFX Connect Real-Time PCR Detection System using low-profile 0.2 ml 8-tube strips. Three pairs of gene-specific primers locating in different gene sites were picked from NCBI for this experiment to reduce errors in detecting from non-specific amplification (Fig 11). F1 and R1 match with NLS-yeGFP, F3 and R3 match with DsRed. Meanwhile, F2 matches with the downstream of NLS-yeGFP and R2 matches with the upstream sequence of DsRed. And we also choose TDH as a reference gene for normalization.
Fig 11. Primers for RT-qPCR
Firstly, the original data of the gene transcription level was determined in a CFX Connect Real-Time PCR Detection System using 8-tube strips (Fig 12). The primary results showed in Cq value indicated there were confirmed differences in transcriptional level of yeGFP-CRISPR-DsRed.
Fig 12. Original data of qPCR
It was clear that using primers F2 and R2, the Cq value was apparently less than using primers F1, R1 and primers F3, R3. This suggests high cleavage efficiency of Csy4 had happened in CRISPR.
In order to display results in a visible and understandable form, we further processed original data. It’s easy to know that many factors may affect gene transcription level of S. cerevisiae, for example, culture time and density of yeast. So, we normalized gene (yeGFP, DsRed and yeGFP-28nt-dsRED) transcriptional level. To be more precisely, Cq values of three gene fragments (yeGFP, DsRed and yeGFP-CRISPR-dsRED) minus the mean Cq Value of TDH gene (mean value is 22.01) separately and calculated each mean values of difference of three gene fragments (Fig 13).
Fig 13. Normalized value of three gene fragments in qPCR
To validate the cleavage efficiency of Csy4, we regarded the transcription level of yeGFP as a background of those three gene fragments considering the transcriptional characteristic of polycistron. Then, we use the Δvalue of those three fragments minus the mean Δvalue of yeGFP gene (mean value = 3.57) separately and calculated each mean values of difference of those three fragments (Fig 14).
Fig 14. Δ(Δvalue) of three gene fragments in qPCR
Based on principle of qPCR which is exponential amplification, we regarded 2 as a base number and the mean values of three fragments as a power to get relatively normalized transcriptional level (Fig 15).
Fig 15. 2-Δ (Δvalue) of three gene fragments in qPCR
Finally, those relatively normalized values were represented as a histogram which is more visible and understandable (Fig 16).
Fig 16. Histogram of qPCR
Based from the relatively normalized results of those three gene fragments, it’s easy to know that the cleavage efficiency of Csy4 is about 98.17%.
4. A strategy for HPLC detection of production with low concentration
BBa_K3544100 and BBa_K3544101 were constructed on the pY26TEF-GDP, and the yield of ABA in S. cerevisiae was estimated to be $141\pm 20\mu g/L$.
While HPLC is a recognized method to detect the concentration of ingredient, lots of iGEM teams, especially those who want to produce natural products heterogeneously, apply HPLC every year. However, the content of products in some project may be low due to limited reaction substrate (in our project, it is FPP), endogenous metabolic interference or inconsistent expression level of enzymes.
When the product concentration is very low, HPLC result could be influenced dramatically by system error of HPLC machine, there are two example:
1. A dinky peak may appear in the time of standard sample peak in negative control, it will be difficult to estimate when product concentration is very low).
2. Showed as followed draft:
Fig 17. The draft figure
Is there any production? Which peak is our production? 5.475 or 5.485? It’s difficult to distinguish.
Meanwhile, standard curve would be unreliable, this is easy to understand.
To solve these problem, we designed a strategy, and process HPLC as follow:
1. Analyze standard sample (10μl, from chromatographic bottle 1. For example, peak time is 5)
2. Analyze negative control (10μl, from chromatographic bottle 2)
3. Analyze negative control (30μl, from chromatographic bottle 2)
4. Analyze experiment group (10μl, from chromatographic bottle 3)
5. Analyze experiment group (30μl, from chromatographic bottle 3)
If product is synthesized successfully, it should fulfill these two conditions:
1. No peak appears very close to time 5, or the peak area is same in step 2 and step3.
2. A peak appears very close to time 5, and the peak area in step5 is almost three times step4.
The yield is calculated by the ratio of peak area (use data from step 1 and step 5).
Our example:
Step 1: Analyze standard sample (10μl), the peak time of ABA in this round of HPLC is 8.985.
Fig 18. HPLC result of 110 mg/L ABA
Step2: Analyze negative control (10μl)，a baseline excursion happened, a minute fluctuation happened in near time 9min.
Fig 19. HPLC result of negative control (10μl)
Step3: Analyze negative control (30μl), no fluctuation happened in near 9min
Fig 20. HPLC result of negative control(30μl)
The experiments of step1-3 and step4-5 are in two batches, and apply another HPLC process of step1, peak time is 8.912.
Fig 21. HPLC result of 110mg/L ABA
Step4: Analyze experiment group (10μl), an apparent peak appears in 8.914, very close to 8.912. Peak area is 7.22.
Fig 22. HPLC result of experiment group (10μl)
Step5: Analyze experiment group (30ul), peak is 19.65 (2.72 times 7.22).
Fig 23. HPLC result of experiment group (30μl)
Calculation:
The peak area of experiment groups (10μl) are $7.02\pm1$.
Relative peak ratio: $5456/7.02= 777.2$, $110mg/777.2=0.141mg$;$110mg/1=0.02mg$.
So the yield is $0.141\pm 0.02mg/L$, $141\pm 20ug/L$.
