Team:Tel-Hai/Parts

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iGEM Tel-Hai 2016

Parts

Part 1 - BBa_K2061000 tGFP ORF under EF1a promoter

This part contains the GFP gene under the human Elongation Factor-1 alpha promoter, which is a constitutive promoter. This part enables the expression of fluorescent protein in mammalian cells. The part was cloned in pSB1C3 between EcoRI and XbaI sites. The reason we did not use the PstI site for cloning is because there are 2 PstI sites within the EF1a promoter sequence and 2 PstI sites inside the tGFP ORF.

The idea is to expose lung epithelial cells to the cross-linked complex (the cholera toxin B subunit and this plasmid) and to look for GFP expression in the cells. This will indicate the insertion of the complex and expression from the plasmid.

PartI 2 -BBa_K2061001 CFTR gene exon 11 – donor part

This part contains the sequence of CFTR gene exon 11 with 400 bp of intron 10 and 340 bp of intron 11 on the flanking regions. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve as a donor sequence for gene editing of ΔF508 mutation in exon 11 of CFTR.

Part 3 - BBa_K2061002 gRNA1 for editing F508 deletion

This part contains 20 bp of target sequence in CFTR exon 11, PAM sequence (NGG) and the gRNA scaffold under the control of U6 promoter and terminator. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve for generating the first gRNA leading the CAS9 to the CFTR exon 11 for gene editing.

Part 4 - BBa_K2061003 gRNA2 for editing F508 deletion

This part contains 20 bp of target sequence in CFTR exon 11, PAM sequence (NGG) and the gRNA scaffold under the control of U6 promoter and terminator. The part was cloned in pSB1C3 between EcoRI and PstI sites. This part will serve for generating the second gRNA leading the CAS9 to the CFTR exon 11 for gene editing.

Part 5 - BBa_K2061005

This part recombinant B-subunit of the E. coli Heat Labile Toxin (LTB) fused via linker sequence to a short viral DNA binding domain (DBD). The part was cloned in pSB1C3 between EcoRI and XbaI sites. This part coded to a protein that can attached to a desired DNA cargo to be deliver to epithelial cell nucleus via endocytosis.