Difference between revisions of "Part:BBa K747020"

Latest revision as of 04:15, 25 August 2020

TAL-Protein_CA2_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.

Here we offer 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.

For a mammalian vector backbone we offer you four plasmids: Eukaryotic TAL expression plasmid, pTALEN, pTAL-TF, pTAL-AID. 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 (Golden Gate Cloning).

BBa K747020; CA2

This Part allows you to predefine the binding affinity of the 4. nucleotide (cytosine) and the 5. nucleotide (adenine) in the 14 nucleotide target sequence.

Target sequence: T NN CA 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. 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. 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. 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 PCR 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

After the restriction-ligation you can transform the cells in your desired E.coli strain. Use LB-Agar plates with kanamycin to select your clones.

Usage and Biology

Gene activation

To show the functionality of our TAL protein as well as the impact of the VP 64 transcription factor fusion protein, we used a TAL-VP64 fusion construct targeting a minimal promotor coupled with the secreted 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 surrounding 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 product absorbs light at 405 nm and can be measured via photometry. This reporter system gives us a couple of advantages over standard EGFP or luciferase systems. First of all, the SEAP is secreted into the cell culture media, therefore we don't have to lyse our cells for measuring, but just take a sample from the supernatant. We are also able to measure one culture multiple times, e.g. at two different points in time. Another advantage is the measurement via photometry which makes the samples quantitively comparable. Interestingly, we did not have to clone a TALE binding site upstream of the minimal promoter (which would be required for other DNA binding proteins) but simply produced a TALE that specifically bound to the given sequence.

Experimental design

---

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.

Activation of transcription

---

To show that our TAL effectors are actually working, we used our completed toolkit to produce a TAL protein which is fused to a VP64 transcription factor. With this TAL-TF construct we targeted a sequence upstream of a minimal promotor that controls transcription of the enzyme secreted alkaline phosphatase (SEAP). In theory, the TAL domain should bring the fused VP64 domain in close proximity to the minimal promotor to activate the transcription of the repoter gene SEAP. The phosphatase is secreted an acummulates in the cell culture media. After 24 and 48 hours, we took samples from the media, stored them at -20°C, and subjected them to photometric analysis.



As it is observable in the graph, co-transfection of cells with TAL and SEAP plasmids(++) yielded a high increase in SEAP activity, compared to the control samples. Also the control experiment with a TAL-VP64 targeting a random sequence shows the specificity of our system. The graph shows the average value of three biological replicates with its standard deviation. We further performed a t-test (Table) to prove if our experiment is statistically significant. As it is clearly observable, the p-values range below a value of 0,05, which indicates that our TAL transcription factor is able to elevate the transcription of the SEAP gene in a statistically significant manner.

After addition of pNPP, the substrate of SEAP, the activity of SEAP was measured over time. In the next image, the results of the first nine minutes of this measurement are shown. After this time, the OD of the double transfection (++) rose too high to be measured by our photometer. As it is clearly visible, the sample with the double transfection shows a profound increase in the OD. This points to the fact that great amounts of SEAP have been secreted into the cell culture media due to elevated gene expression. In the other samples almost no SEAP activity was measureable. The sample transfected with only the SEAP plasmid showed the highest OD but this effect was not statistically significant (p-value:0,25/0,51).
In the samples that had been taken 48h after double transfection, the same effects could be demonstrated.
Furthermore, we reapeated the same experiment for a second time. The corresponding data can be viewed here:

Second Experiment.

=

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 36
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 165
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 168
  

Functional Parameters: Austin_UTexas

BBa_K747020 parameters

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.