Team:FAU Erlangen/Tour41
Part Documentation
TALE
TALEs (Transcription-Activator-Like Effectors) are a family of effector molecules that consist of two constant domains and a central domain made of 17 repeats of 34 amino acids with two hypervariable positions, called Repeat-Variable Di-residues or RVDs. TAL effectors were found to be able to induce gene expression in plants by binding a specific sequence in the promoter region. By deciphering the code of TAL specificity it became possible to create new TAL effectors binding specific, purposefully targeted DNA sequences.
TALE1 / BBa_K1861050
The first TALE binds the sequence T CTCCACCCCATT , has a SpyTag fused to its N-terminus and has been cloned into our expression cassette before submitting.
TALE2 / BBa_K1861060The second TALE binds the sequence T GTCGTAACAACT, has a SpyTag fused to its N-terminus, and has been cloned into our expression cassette before submitting.
TALE3 / BBa_K1861070The third TALE binds the sequence T CCGACTCGCTGT, has a SpyTag fused to its N-terminus, and has been cloned into our expression cassette before submitting.
TALE CD / BBa_K1861020The constant domain can be used to clone sequence-specific TALE domains into it by utilizing the Golden Gate method and has been cloned into our expression cassette before submitting.
EC (Expression Cassette) / BBa_K1861010
This cassette includes an rpd3 promoter, the start codon with a His-Tag, a spacer that can be cut out with BamHI and SacI, the stop codon and an adh1 terminator. This cassette is meant to be used for the expression of genes in S. cerevisiae.
Rpd3 / BBa_K1861040
Rpd3 is a yeast (Saccharomyces cerevisiae) endogene histone deacetylase, which shares 60% sequence identity with the human deacetylase. The deacetylating effect of rpd3 is very local, it occurs about 150-250 bp from the target sequence, which is about the distance covered by one histone.
The rpd3 we used was fused C-terminally to a SpyCatcher and has been cloned into our expression cassette before submitting.
YFP / BBa_K1861030
The target cassette was designed as a sequence carrying the promoter-region of the CMV-Promoter as well as the target sequence T GTCGTAACAACT, which is not found in yeast strains (K699 as well as BY4742). For a ribosome binding site, the Kozak sequence Bba_K792001 was chosen and inserted between the last base of the promoter and the start codon ATG. Right behind the start codon and right before the terminator BBa_J63002, a XhoI-restriction site and a SalI restriction site, respectively, were introduced. A 44bp long spacer between them allowed us to clone the YFP insert into the cassette after we added the above cloning sites to the termini of the insert. On the edges of the contstruct the sequences for an insertation in the his3 sequence via homologue recombination are included.