We improved the GFP part. In the beginning, we directly took the GFP part into practice, but it is observed that GFP has a long half-life, which is not convenient to further measurement. So, we designed and constructed the nfGFP part(new GFP which degrades fast) with a shorter half-life to improve the feasibility.
Measurement of nfGFP half-life
1. Characterization part design
Ubiquitin is a small protein founded in all eukaryotes whose primary function is to label a protein to be quickly recognized and degraded by the proteasome in the cell. We added a ubiquitin tag before the GFP structural gene, predicting that this would result in faster recognition and degradation of the expressed ubiquitin-tagged-GFP fusion protein by the ubiquitin degradation mechanism. To test our hypothesis, we designed the following parts and experiments.
Figure 1. The sketch map of the characterization plasmid of nfGFP
To avoid misunderstanding, we show the diffenences between two nfGFP parts in our project. When measuring the degradation rate of nfGFP, we used the inducible promoter Pgal and expressed it on a plasmid vector. In contrast, when verifying chromosome-free cell formation with nfGFP, the promoter is TDH3 which can express frequently, and we integrated it on the chromosome for expression by homologous recombination.
2. Experiment design
Experimental material: S. Cerevisiae with a nfGFP expression plasmid
Before the induction, the cells were cultured and enriched in the medium for 15 hours. We measured the OD600nm values and the nfGFP fluorescence values. When the nfGFP fluorescence signals reached a steady value, we stopped inducing, then changed cells to standard medium. Considering the end of induction as time zero, we recorded the changes of OD600nm and nfGFP fluorescence signals every two hours. Due to the shutdown of nfGFP expression in the cell, the cells can no longer produce reporter proteins, so the change in fluorescence signal intensity since then roughly reflects the degradation rate of nfGFP. By measuring the nfGFP fluorescence values, we plotted the degradation curves of nfGFP and calculated the half-life of nfGFP.
3. Experiment operation
(1) Enrichment and induction: Due to a large amount of culture medium required for subsequent measurements, we used shake flasks with 25 ml of induction medium to minimize the effect of the volume changes of the culture medium.
(2) Measurement of OD600nm: We used a UV spectrophotometry to measure OD600nm. To ensure data reliability, we unified the dilution multipliers while ensuring that the values are between 0.1-1.0 and as far as possible between 0.2-0.8.
(3) Measurement of fluorescence value: We use a Microplate Reader to measure the nfGFP fluorescence value. After the OD600nm measurement, we wash off the culture medium and resuspend the cells with sterile water. Then, add 200 μl of the suspension to be measured to each well of the 96 well microtiter plate. We set blank reference (water), control groups and experimental groups in three parallel groups when adding samples. The excitation/emission light wavelength of nfGFP in the Microplate Reader was 488/535 nm.
4. Data analysis and processing
Since the sample is diluted, the fluorescence intensity is equal to the measured fluorescence intensity multiplied by the dilution factor. Besides, the measured fluorescence intensity value should also subtract the blank value. In summary, the formula for calculating the nfGFP fluorescence intensity should be as follows.
V':the true fluorescence intensity of nfGFP/200μl sample
V: the measured value of nfGFP fluorescence signal/200μl sample
C: blank reference(water)
ω: the dilution factor of sample
The values of the three parallel groups of samples were averaged and plotted, the calculated data are shown in the figure. We use the following formula to fit the measured value and calculate the half-life value.
N: fluorescence measurement over time
N0: initial fluorescence value
τ: half life value
Figure 2. The degradation curve of nfGFP
5. Results analysis
From the results, it is observed that: When we stop inducing, the nfGFP signal decreased fast, indicating that nfGFP was rapidly degraded in the cells after the gene was no longer expressed.
The data show that the half-life of nfGFP is around 7.558h, which is significantly reduced compared to the half-life of GFP(33h), confirming the validity of our design.
 John R. Houser, Eintou Ford, Sudeshna M. Chatterjea. (2012). An improved short‐lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae. Yeast(12), 519-530.