Difference between revisions of "Part:BBa K747002"

Revision as of 18:08, 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.

As mammalian vector backbone we over you three plasmids: Bba_K747096 to Bba_K747098. You can clone your Direpeats directly between the first and the fourteenth repeat.

The position of the Direpeat inside a TAL-Protein is determined by the sticky-end, which will be produced, if you digest the Direpeats with the type II restriction enzyme BSmBI. The Cloning can be performed in one single restriction-digestion.

BBa K747002; AG1

This Part allows you to predefine the binding affinity of the 2. nucleotide (adenine) and the 3. nucleotide (guanine) in the 14 nucleotide target sequence.

Target sequence: T AG NN NN NN NN NN T

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).

To ligate your own TAL-Protein, 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

Usage and Biology

To show the functionality of our TAL protein as well as the impact of a VP 64 transcription factor fusion protein, we used a TAL-VP64 fusion construct targetting a minimal promotor coupled with the secreated alkaline phosphatase (SEAP). The product of the reporter gene SEAP is as the name tells a phosphatase that is secreted by the cells into the surrunding media. The existence of SEAP and therefore the activity of the promotor can be measured by the addition of para-Nitrophenylphosphate (pNPP). The SEAP enzyme catalyzes the reaction from pNPP to para-Nitrophenol, this new formed product absorps light at 405 nm and can be measured via photometer. This reporter system gives us a couple of advantages over standard egfp or luciferase systems. First of all the SEAP is secreated into the cell culture media, therfore we dont have to destroy our cells to meaure but just take a sample from the supernatant. Also we are able to measure one culture multiple times for example at two different time points. Another advantage is the measurement via photometer which makes the samples quantitive compareable.

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The experiment was done with four different transfections, either no plasmid, only the TAL vector, only the SEAP plasmid or a cotransfection of both plasmids. The cells were seeded on a twelve well plate the day before in 500µl culture media per well. The transfection was done with CaCl2 after a cellculture course protocol witten by the lab of professor Weber.

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To show that our TAL effectors are actually working, we used our finished toolkit to produce a TAL coupled to a VP64 transcriptionfactor. With the TAL we targeted a region in upstream of a minimal promotor coupled secreted alkaline phosphatse gene (SEAP). In theory the TAL domain should bring the coupled VP64 domain in close proximity to the minimal promotor an activate the transcription of the repoter gene SEAP. The phosphatase is secreated an acummilates in the cell culture media. After 24 and 48 hours we took samples from the media kept them at -20°C and analyzed them two days later with an photometer. (For detailed experiment design and theory of SEAP look at our "Experiment" page in the project section.)







As you can see in the graph we were able to demonstrate a high increase in SEAP activity compared to the control samples and the co-transfection of TAL and SEAP plasmids (++). The graph shows the average of three biological replicates with its standard deviation. In the next table we did a ttest to prove the statistical significance, the yellow highlighted fields are the p-values for our double transfections. As you can see they are clearly below a p-value of 0,05 which demonstrates their statistical significance.

The next image shows the SEAP measurement over the first nine minutes, after this time the OD of the double transfection (++) got to high to be measured by our photometer. As you can see the OD of the double transfection rose very fast indicating a high amount of SEAP reacting with the substrate pNPP. In the other samples no SEAP was measureable, the sample transfected with only the SEAP plasmid showed the highest OD but not statisticaly significant (p-value:0,25/0,51). The results for the samples taken after 48 hours showed the same behavior.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]