Part:BBa_K747099

Eukaryotic TAL expression plasmid

All ccdb parts have been withdrawn from the Registry. Samples of parts containing the ccdB gene cannot be requested. - iGEM HQ

Once the MammoBrick was ready, we inserted the TAL open reading frame and thereby evaluated, how easy it would be for future iGEM students to insert any desired ORF in eukaryotic cells.

Designing the TAL open reading frame:
For this purpose, we designed a TAL ORF by adding the following modifications to the TAL open reading frame in [http://www.everyvector.com/sequences/show_public/18260 pTALEN_(v2)_NG]:

1. We removed all EcoRI, XbaI, SpeI, PstI, BsmBI, BbsI and PmeI restriction sites.
2. We replaced the BsaI restriction sites for inserting direpeats by BsmBI sites, because – according to the manufacturer - BsmBI is better suited for digest over one hour.
3. We added a consensus RBS in front of the ORF for expression in bacteria
4. We added a His-Tag to the n-terminal end to allow protein purification.
5. We flanked the whole sequence with the iGEM prefix and suffix.
6. Most importantly, we replaced the FokI nuclease at the C-terminal end of the protein by one of our inventions: The Plug and Play Effector Cassette.

This whole construct was synthesized by IDT.

Plug and Play Effecor Cassette: Our project was designed to enable future iGEM teams to easily use the powerful TALE technology. On top of that, we wanted to built a TALE platform which allows iGEM students to freely develop their own TAL constructs. We therefore invented the easy-to-use Plug and Play Effector Cassette (PPEC), which can be used to fuse BioBricks, that are in the [http://2012.igem.org/Team:Freiburg/Project/Golden Golden Gate standard], to the c-terminus of the TAL protein.

The PPEC consists of two BbsI binding sites that point in opposite directions. Digestion with BbsI leads to removal of the PPEC and to the formation of sticky ends at which the upstream sticky end (GGCA) are the last 4 nucleotides of the tal protein and the downstream sticky end (TAAA) contains the stop codon. When an equimolar amount of the effector containing plasmid (flanked also by BbsI sites and the same overlaps) is added to the GGC mix, the effector is cut out of the iGEM vector and ligated into the eukaryotic TAL expression vector in-frame and without a scar. We have optimized this reaction by systematically testing different reaction buffers and thermocycler programs and came up with the following protocol:

Component	Amount (μl)
BpiI/BbsI (15 U)	0,75
T4 Ligase (400 U)	1
DTT (10 mM)	1
ATP (10 mM)	11,5
G-Buffer (10x, Fermentas)	1
parts	40 fmoles each
ddH2O	Fill up to 10
Total	10

Thermocycler programm:
1.     37°C, 5 min
2.     20 °C, 5 min
go back to 1. 20 times
4.     50°C, 10 min
5.     80°C, 10 min
5.     4°C, ∞

But even Golden Gate cloning is not 100 % efficient. In order to remove those plasmids that did not take up a vector insert, we added the restriction site of the blunt end cutter PmeI (MssI) to the PPEC. We chose PmeI because it has a 8 bp binding site, which is very unlikely to occur in the gene of an effector that you would like to fuse with the TAL gene. So after performing the Golden Gate reaction described above, we digested with MssI fast digest (fermentas) according to the following protocol.

Component	Amount (μl)
GGC-Product	10
PmeI/MssI FastDigest	1
Fast Digest Buffer (10x)	1,5
ddH2O	2,5
Total	15

Thermocycler programm:
1.     37°C, 1h
2.     80 °C, 20 min
5.     4°C, ∞

This linearizes all vectors that do not contain the effector (at least, we do not see colonies on the negative control plate). To be sure, these linearized vectors do not religate, perform the following digest with T5 exonuclease, which specifically removes linearized DNA:

Component	Amount (μl)
Product of PmeI digest	7,5
T5 Exonuclease	1
Total	8,5

Thermocycler programm:
1.     37°C, 1h
2.     80 °C, 20 min
5.     4°C, ∞

Usage and Biology

Gene activation

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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

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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.

Activation of transcription

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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
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 966
  Illegal suffix found in sequence at 3901
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 966
  Illegal SpeI site found at 3902
  Illegal PstI site found at 3916
  Illegal NotI site found at 972
  Illegal NotI site found at 2146
  Illegal NotI site found at 3909
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 966
  Illegal BglII site found at 1563
  Illegal BglII site found at 2914
  Illegal BamHI site found at 2217
  Illegal BamHI site found at 2920
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 966
  Illegal suffix found in sequence at 3902
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 966
  Illegal XbaI site found at 981
  Illegal SpeI site found at 3902
  Illegal PstI site found at 3916
  
• 1000
  COMPATIBLE WITH RFC[1000]

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