Team:SUSTech Shenzhen/Contribution

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Contribution

SUSTech_Shenzhen 2019 iGEM team have contributed the characterization of part BBa_K1537016 by linked the 2A sequence with two target protein (cytokine IL-10 and eGFP, enhanced green fluorescent protein) and measured the expression level of the desired protein. By the measurement data, we conformed the feasibility of 2A peptide in a new chassis (HeLa cell). We also provided quantitative data to analyze the influence of 2A's cleavage function on the expression level of downstream protein.

First, we obtained the DNA sequence of 2A peptide from 2019 iGEM distribution and linked our target protein sequence to construct two plasmid: 5xUAS-EGFP and 5xUAS-IL10-2A-EGFP, 5xUAS is the binding site of part BBa_K2986003(GAVPO, a light-switchable trans activator). (figure1-2: plasmid construction)

Figure 1: eGFP plasmid construction
Figure1: eGFP plasmid construction
Figure2: IL10-2A-eGFP plasmid construction
Figure2: IL10-2A-eGFP plasmid construction
Then, we transfect the plasmid sequence and the BBa_K2986003 into HeLa cell line, and measure the induced protein expression level. We used flow cytometry to measure the eGFP expression level, and ELISA (enzyme linked immunosorbent assay) method to measure the IL-10 expression level. (figure3-4：flow cytometry of eGFP expression level) (figure4-5: ELISA test of IL-10)

Figure 3: Flow cytometry of eGFP expression level of two cell lines
Figure 3: Flow cytometry of eGFP expression level of two cell lines
Figure 4: Comparison of eGFP expression level of two cell lines (with our without 2A sequence)
Figure 4: Comparison of eGFP expression level of two cell lines (with our without 2A sequence)
Figure 5: IL10 ELISA test
Figure 5: IL10 ELISA test
Figure 6: Result of IL10 ELISA test
Figure 6: Result of IL10 ELISA test


From figure 3-6, we conformed that this part is functional in HeLa cell line: the IL-10 and eGFP is cleaved by 2A peptide and successfully expressed. Our quantitative data from figure 3-4 also provided another characterization feature of 2A peptide: expression level of the latter protein linked after 2A (eGFP level in the IL10-2A-eGFP cell line) is lower than the control group without 2A (eGFP cell line). The eGFP expression level after 2A is decreased 29.64%.