Part:BBa_K1537017

GSG linker+T2A

T2A

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)

[http://2019.igem.org/Team:AFCM-Egypt# Team:AFCM-Egypt 2019] have worked to create an improved Glycine serine linker in part BBa_K1537017 in our new part BBa_K3244026 to adapt for our bispecific extracellular domain which should carry 2 scFv targeting 2 different antigens. This part has been modified through codon optimization -which has represented an issue for our fragment DNA synthesis due to the need for repetition of linker sequence between 4 different chains of 2 scFvs- and amino acid sequence length editing t(GGGGS)5 to act as an inter-chain linker between the 2 scFv for enhanced production and stability of the bispecific scFv. T2A was also added as a basic part in BBa_K3244020. For results of functional characterization please refer to our parts Page EGYPT-AFCM 2019 Parts Significant difference between GSSS_ BBa_K1537017 engineered and GSSS_BBa_K3244026 T-cells(p<0.05), higher expression levels were observed in GSSS_BBa_K3244026 group.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]