Difference between revisions of "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

The first TALE binds the sequence T CCGACTCGCTGT, has a SpyTag fused to its N-terminus and has been cloned into our expression cassette before submitting.

TALE2

The second TALE binds the sequence T CTCCACCCCATT, has a SpyTag fused to its N-terminus, and has been cloned into our expression cassette before submitting.

TALE3

The third TALE binds the sequence T GTCGTAACAACT, has a SpyTag fused to its N-terminus, and has been cloned into our expression cassette before submitting.

TALE CD

The 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)

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

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

This part contains eYFP downstream of a CMV-Promoter. Upstream of the startcodon an registry Kozak sequence Bba_K792001 is located. Transskriptional termination is achieved through adh1 terminator BBa_J63002. On the edges of the contstruct the sequences for an insertation in the his3 sequence via homologue recombination are included. The eYFP-sequence is exchangeable with a SalI-XhoI digest.