Difference between revisions of "Part:BBa K3939111"

Revision as of 12:43, 20 October 2021

Fast degrading GFP

The ubiquitin tag is fused to the N-terminal of the GFP to make the GFP a rapidly degrading GFP

Sequence and Features

We improved the GFP part. In the beginning, we directly took the GFP part into practice, while later, it is observed that GFP has a relatively long half-life, which is not conducive to further measurement. This section we will illustrate how we test our improvel designs correspondingly, showing the reasonability in the process and the reliability of the results.

1. Characterization part design:

Ubiquitin is a small protein found 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.

Fig.1 nfGFP Schematic (plasmid vector 、Pgal、Ubiquitin tag、nfGFP、terminator)

To avoid misunderstanding, two nfGFP parts in the project are different. 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 was the frequently expressed promoter TDH3, and we integrated it on the chromosome for expression by homologous recombination.

2. Experiment design:

Control group: S. Cerevisiae with a normal GFP expression plasmid
Experiment design:
Control group: S. Cerevisiae with a regular GFP expression plasmid
Experimental group: S. Cerevisiae with an nfGFP expression plasmid

Before the induction, the cells in the experimental group were cultured and enriched in the medium for 15 hours. Considering the start of induction as time zero, we measured the OD600 values and nfGFP fluorescence intensity value of the cells every two hours. When the nfGFP fluorescence signals of the experimental groups reached a steady value, we stopped the induction, then changed to standard medium, and recorded the changes of OD600 and GFP fluorescence signals every two hours. Due to the shutdown of GFP expression in the cell, the cell can no longer produce reporter protein, so the change in fluorescence signal intensity since then roughly reflects the rate of degradation of both parts. By measuring the OD600 values and nfGFP fluorescence values, we plotted the degradation curves of the two parts and compared them.

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 changes in culture medium volume.
(2) Measurement of OD600: We used a UV spectrophotometer to measure OD600. 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 an enzyme-labeled instrument to measure the nfGFP fluorescence value. After the OD600 measurement, wash off the culture medium and resuspend the cells with sterile water. Add 200 μl of the suspension to be measured to each well of the plate of the enzyme-labeled instrument. We set blank reference (water), control, and experimental groups in three parallel groups when adding samples. The excitation/emission light wavelength of nfGFP in the enzyme-labeled instrument was 488/535 nm.

4. How we analyze and process data

The values of the three parallel groups of samples were averaged and plotted.
In our experiments, we found that the fluorescence signal has a linear relationship with OD600 when the OD600 is less than 1, which means we should control the OD600 of the sample for measuring the fluorescence value to be less than 0.8.
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

V^'=ω*(V-C)

V^':the fluorescence intensity of nfGFP
V: the measured value of nfGFP fluorescence signal in experimental group
C: blank reference
ω：sample dilution factor

The corrected values are shown in the figure：

After cessation of induction, the nfGFP signal decreased to the level when the expression was not induced, indicating that nfGFP was rapidly degraded in the cells after the nfGFP 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 time of GFP on average, confirming the validity of our design.