Team:NYC B1O/Contribution

Team Attributions
Description Contribution Engineering Implementation Wetlab Notebook
Model
Human Practices Collaborations

Contribution:


Our use of eGFP optimized for expression in mammalian cells can be extended to use by other iGEM teams investigating diseases that affect humans or other mammals requiring the tagging and subsequent visualization of proteins. We can contribute to other teams by uploading this specific sequence. We can also contribute to other iGEM teams by uploading the sequences of wild-type alpha-synuclein, allowing other teams to investigate solutions to Parkinson’s disease. We incorporated part BBa_K2619011 in our alpha-synuclein construct.

After using part BBa_K2619011 from the iGEM registry, we contributed to it by adding more informations in the main page regarding the structure and function of human HSP70 chaperone (see highlighted text in the first image below). We also added more information regarding the parameter of this part (see second image below).




pEGFP-alphasynuclein-WT

This plasmid contains the coding sequence of wild type alpha-synuclein (aSYN) attached to the green fluorescent protein (EGFP). The expression of EGFP-aSYN is under control of the constitutive strong CMV promoter, allowing for the expression of EGFP-aSYN in all mammalian cells. EGFP and aSYN are connected by a flexible linker that allows for their independent folding into their expected 3D conformation. This plasmid was used to extract the coding sequence of aSYN along with the flexible linker by PCR in order to build pHSP70-alpha-synuclein-WT-P2A-mCherry and pHyD-LIR(p62)-alpha-synuclein-WT-P2A-mCherry by Gibson assembly.

pEGFP-HSP70

This plasmid contains the coding sequence of HSP70 attached to EGFP. The expression of EGFP-HSP70 is under control of the constitutive strong CMV promoter, allowing for the expression of EGFP-HSP70 in all mammalian cells. EGFP and HSP70 are connected by a flexible linker that allows for their independent folding into their expected 3D conformation. This plasmid was used to extract the coding sequence of HSP70 by PCR in order to build pHSP70-alpha-synuclein-WT-P2A-mCherry by Gibson assembly.

HyD-LIR(p62)

Synthetic oligonucleotide containing the coding sequence for the autophagy tag HyD-LIR(p62). This synthetic oligonucleotide was designed in silico in order to build pHyD-LIR(p62)-alpha-synuclein-WT-P2A-mCherry by Gibson assembly.