Difference between revisions of "Part:BBa K1537016"

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−	<img src="https://2019.igem.org/wiki/images/6/6b/T--SUSTech_Shenzhen--p2Aflow.png" alt="Figure 1: eGFP plasmid construction" style="width:100%;" />	+	<img src="https://2019.igem.org/wiki/images/6/6b/T--SUSTech_Shenzhen--p2Aflow.png" alt="Figure 1: eGFP plasmid construction" style="width:70%;" />
	<figcaption>Figure1: eGFP plasmid construction</figcaption>		<figcaption>Figure1: eGFP plasmid construction</figcaption>
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Revision as of 17:36, 19 October 2019

GSG linker+P2A

P2A

2A peptide sequences were found in Picornaviruses to mediate "cleavage" between two proteins. We use 2A peptide-linked multicistronic vectors to express multiple proteins from a single open reading frame (ORF) effectively. To minimize the risk of homologous recombination, it is important to use different 2A peptide sequences if more than two genes are being linked. The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammalian cells, yeast, and plants.(A El et al，2004)

GSG linker

GSG linker is an oligopeptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage.( Andrea L et al，2012)

Characterization done by SUSTech_Shenzhen 2019

SUSTech_Shenzhen 2019 iGEM team improved the characterization of this part 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)

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)

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%.

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References

Abdelhak El Amrani,Abdellah Barakate,Barak M. Askari, Xuejun Li, Alison G. Roberts,Martin D. Ryan, and Claire Halpin.(2004)Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene.Plant Physiology.Vol. 135, pp. 16–24

Andrea L. Szymczak-Workman, Kate M. Vignali, and Dario A.A. Vignali.(2012)Design and construction of 2A vectors.Cold Spring Harb Protoc :199-204.

Sequence and Features