Team:NJTech China/Results
1. Construction and validation of murine IL-2-RFP fusion protein and IL-2 gene expression cassette
The plasmid pGAP-ss-IL-2-RFP was extracted using AXYGEN AxyPrep Plasmid Miniprep Kit, and the extracted plasmid was digested by BamHⅠ single digestion. The enzyme digestion result was verified by agarose gel electrophoresis.
The coding gene of the signal peptide and interleukin 2 was amplified by using the recombinant plasmid pGAP-ss-IL-2-RFP as template and ss-IL2 F and ss-IL2 R as primers.
The plasmid pGAP-KanR was extracted using AXYGEN AxyPrep Plasmid Miniprep Kit, and the extracted plasmid was digested by XbaⅠ single digestion. The enzyme digestion result was verified by agarose gel electrophoresis.
2. Transformation and verification of murine IL-2-RFP fusion protein and IL-2 gene expression cassette
The recombinant plasmid pGAP-ss-IL-2 was linearized by BamHⅠ single digestion. The enzyme digestion result was verified by agarose gel electrophoresis.
Pick a single colony growing on the resistant plate and transfer to a new YPD plate. Colonies growing on the transfer point plate were picked for colony PCR to select positive transformants.
For the positive clones identified by colony PCR, the yeast genome was extracted using the Solarbio Yeast Genomic DNA Extraction Kit. Using the proposed genome as a template and ss-IL2 F and ss-IL2 R as primers, genomic PCR was performed to verify whether our target gene was successfully integrated into the yeast genome.
3. Expression, collection and purification of our target proteins
Four days' microscopic examination results showed that all the fermentation broths were not contaminated with bacteria during our expression process.
The '1, 2, 3, 4' in the legend represents four flasks for fermentation. It can be seen from the line graph that at the beginning of fermentation, with the prolongation of expression time, the OD600 value shows a gradually increasing trend. When the value stabilizes, we conclude that the expression process is over. The collection and purification of the product can be carried out in the next step.
Red fluorescent spots were observed under fluorescent illumination at a wavelength of 515-520 nm. However, yeast cells cannot be found in the visible field of view.
We further confirmed the presence of the target protein by SDS-PAGE.
Interpretation of experimental results:
In the black box line is the protein gel of the 100-fold diluted sample. The samples from left to right were centrifuged supernatant, ultrafiltration supernatant, and ultrafiltration. No protein bands appeared in the undiluted sample and the 100-fold diluted sample, except that the 100-fold diluted ultrafiltration showed a protein band of approximately 40 kDa.
Analysis：
In theory, the centrifugation supernatant (i.e. the first strip) should have multiple strips, and at least one strip should appear in the ultrafiltration supernatant or ultrafiltration. Moreover, since the membrane used for ultrafiltration is 10 kDa, ultrafiltration should not reveal a faint 40 kDa protein band. Therefore, the above results have yet to be verified.
After reprocessing the sample, we performed SDS-PAGE again. In order to rule out the impact of non-standardized individual experimentation on the results, we invited a graduate student in the lab to help us with this experimental operation. The results showed that there were still no bands in the three samples. The picture above shows the photo of the SDS-PAGE gel. Among them, the left side is the protein marker lane, the middle is the sample lane, and the far right is the sample strip of a graduate student in the laboratory that day who shared the gel with us that day.
Analysis：
Combined with the previous experimental results, we speculated that the protein was not dissolved in the fermentation broth, so no bands appeared on the SDS-PAGE gel these days. And that faint strip is most likely a hybrid protein.
In summary, there may be two cases: 1 protein may be precipitated and mixed with yeast cells in the form of a precipitate during centrifugation. 2 The protein may still be intracellular, which also explains the results of fluorescence microscopy. The red spot that is seen may be the hybrid protein released by the broken yeast.
Validation Results
Figure 1. In vitro release profiles of BSA with different concentrations HA/γ-Glu hydrogels at 37 °C. A indicates 12% (ωt) γ-Glu and 12% (ωt) HA cross-linking hydrogels. B indicates 10% (ωt) γ-Glu and 10% (ωt) HA cross-linking hydrogels.
Fig. 1 shows the BSA-loaded hydrogels release kinetics. The release curves were fitted by the software Origin 8.0 according to several kinetic models. The drug showed a continuous release state in two kinds of cross-linking hydrogels. Compared with the release time of drugs from injectable HA hydrogels prepared by physical mixing (about one hours), the release of BSA from HA/γ-PGA hydrogels with various compositions showed sustained release behavior (>80 h). The crosslinking density is one of the factors that affect drug release, so the factors influencing the hydrogel crosslinking degree also affect the drug release. Next, we load different concentrations BSA and extend the measurement time to see an obvious drug releasing trend.
In summary
After all the above experiments were completed, we discussed all the problems that occurred and carefully recalled the entire process of designing the experiment. Finally, the designer of the sequence found that an extra signal peptide was hidden in the front of our IL-2 sequence, which may be used to mediate the secretion of IL-2 by T cells. However, this signal peptide is superfluous to our yeast and may affect the conformation of the expressed protein, which in turn affects the function of the protein.
During this time, our previous animal model team members received a response from the iGEM Safety Committee on animal experiment applications. Since it was the first time for our team members to design their own animal experiments, many points were not perfect enough. Although the experimental ethics approval and 3R report have been obtained, the feedback from the Safety Committee indicates that there is still much room for improvement in the experimental design.
As students majoring in Bioengineering and Pharmacy, we all understand the many ethical issues that animal experiments face and the inevitable harm to those animals. And iGEM, as an international synthetic biology academic event, does not want us to do unnecessary damage to life. Finally, we decided to give up the animal experiment and design more cytology experiments, trying to prove our experiment results in another way.