Difference between revisions of "Part:BBa K747002"

Revision as of 14:53, 25 September 2012

TAL-Protein_AG1_Direpeat

TAL- Proteins

This part can be used to synthesize a 14 nucleotide Transactivator-like (TAL) protein i.e. DNA-binding proteins. In contrast to zinc-finger proteins they consist of domain-like repeats in their primary structure that differ only by two amino acids. The two aminoacids determine the nucleotide they bind.

Her we over a plasmid set of 96 Direpeats (Bba_K747000 to Bba_K747095) which allows you to assemble your own specific nucleotide target sequence. Every Direpeat is able to bind two specific nucleotides and is determined by his position in the 14 repeating Domains.

Designing a TAL-Protein:

To build a functional Tal- Protein, you have to choose a 14 nucleotide target sequence. Be aware that the first and the fourteenth nucleotide of the sequence has to be a Thymin. The second to the thirteenth nucleotide can be determined by choosing six DiRepeats of the plasmid set:

• Bba_K747000 to Bba_K747015 predefines the nucleotide of the 2. and the 3. nucleotide.
• Bba_K747016 to Bba_K747031 predefines the nucleotide of the 4. and the 5. nucleotide.
• Bba_K747032 to Bba_K747047 predefines the nucleotide of the 6. and the 7. nucleotide.
• Bba_K747048 to Bba_K747063 predefines the nucleotide of the 8. and the 9. nucleotide.
• Bba_K747064 to Bba_K747079 predefines the nucleotide of the 10. and the 11. nucleotide.
• Bba_K747080 to Bba_K747095 predefines the nucleotide of the 12. and the 13. nucleotide.

Golden Gate Cloning

Golden Gate Cloning exploits the ability of type IIs restriction enzymes (such as BsaI, BsmBI or BbsI) to produce 4 bp sticky ends right next to their binding sites, irrespective of the adjacent nucleotide sequence. Importantly, binding sites of type IIs restriction enzymes are not palindromic and therefore are oriented towards the cutting site (indicated by the arrows in figure 2 restriction site). So, if a part is flanked by 4 bp overlaps and two binding sites of a type IIs restriction enzyme, which are oriented towards the centre of the part, digestion will lead to predefined sticky ends at each side of the part. In case multiple parts are designed this way and overlaps at both ends of the parts are chosen carefully, the parts align in a predefined order (figure 3). In case a destination vector is added, that contains type IIs restriction sites pointing in opposite directions, the intermediate piece gets replaced by the assembled parts – magic! After transformation, the antibiotic resistance of the destination vector selects for the right clones. Golden Gate Cloning is typically performed as an all-in-one-pot reaction. This means that all DNA parts, the type IIs restriction enzyme and a ligase are mixed in a PCR tube and put into a thermocycler. By cycling back and forth 10 to 50 times between 37°C and 20°C, the DNA parts get digested and ligated over and over again. Digested DNA fragments are either religated into their plasmids or get assembled with other parts as described above. Since assembled parts lack restriction sites for the type IIs enzyme, the parts get “trapped” in the desired construct. This is the reason why Golden Gate Cloning assembles DNA fragments with such exceptional efficiency. We successfully used this approach to assemble whole TAL effectors vector from six different parts and cloned it into an expression vector – all in one reaction (see below).

Pipette the following volumes in one tube:

Use the following protocol to incubate the restriction-ligation mixture:

• 37°C for 5 min and 30°C for 5 min (repeat this Step 13 times)
• 50°C for 10 min
• 80°C for 10 min
• store at 4°C

Sequence and Features