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<li><a href=''><span>Team members</span></a></li>
<li><a href=''><span>Team members</span></a></li>
<li><a href=''><span>Attributions</span></a></li>
<li><a href=''><span>Attributions</span></a></li>
<li><a href=''><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" width="25px" src=""></img></td></tr></table></a></li>
<li><a href=''><span>Gallery</span></a></li>  
<li><a href=''><span>Gallery</span></a></li>  
<li><a href=''><span>Sponsors</span></a></li>  
<li><a href=''><span>Sponsors</span></a></li>  

Revision as of 21:50, 25 October 2012

TAL-based transcriptional regulators

We created several TAL repressors by fusions of the KRAB repression domain to different positions of the TAL DNA-binding domains and reporter plasmids with their respective binding sites.

We created several TAL activator constructs by C-terminal fusion of the VP16 domain with TAL DNA-binding domains and reporter plasmids with their respective binding sites.

We improved and characterised the NicTAL DNA-binding domain (deposited by the iGEM2010 team Slovenia) by adding a missing subdomain of the protein and created a designed repressor.

TAL:KRAB fusions exhibited over 90% repression of reporter gene expression regardless of the position of the KRAB domain.

Minimal promoters used for construction of reporter plasmids showed no or minimal leakiness and were activated over 1500-fold by TAL:VP16 fusions

Our experimental results on designed TAL regulators provided parameters for the quantitative deterministic modeling of bistable switches.

Designed TAL transcriptional regulators

For the past two decades, engineered zinc finger proteins have been extensively used for targeting specific DNA sequences. However, in spite of the many years of technological development, engineered zinc finger proteins are not able to target every desired DNA sequence due to the impact of neighbouring fingers on the recognition of base pairs. Recently DNA-binding proteins with a simpler DNA recognition code were discovered. Transcription activator like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain (Figure 1A). A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). As evident from the crystal structure of TAL effectors (Mak et al., 2012; Deng et al., 2012; Figuer 1B), all TAL repeats have almost identical conformations, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.

Figure 2. Models of 3D structures of TAL DNA-binding domains fused with the KRAB repression domain (A) and VP16 activation domain (B).

TAL effectors have mostly been used as a tool for plant or mammalian genome editing. The basic idea is the same as with zinc finger nucleases, with TALs replacing zinc fingers as the specific DNA-binding domain (Miller et al., 2010). Several groups (Miller et al., 2011; Zhang et al, 2011) have also designed TAL effectors for specific gene activation, by fusing them with either the Herpes simplex virus VP16 activation domain or its tetrameric derivative VP64 (Figure 2). After we already initiated the iGEM 2012 project, TAL repressors were reported by Garg et al., who created TAL effectors fused with the KRAB transcriptional repression domain.


Figure 3. Schematic representation of the tested plasmids. (A) TAL repressors; fusions of TAL DNA-binding domains with the KRAB repression domain. (B) TAL activator; fusion of TAL DNA-binding domain with the VP16 activation domain. Expression of TAL effectors is under the control of constitutive CMV promoter. (C) Reporter plasmids used to test efficiency of TAL regulators. TAL DNA-binding sites are placed upstream of either a CMV promoter (repression) or a minimal promoter (activation), driving the expression of reporter genes (firefly luciferase or mCitrine).

The TAL transcriptional activators and repressors were basic tools in our iGEM project. We designed and characterised three functional TAL regulators (TALA, TALB and TALD) by fusing TAL DNA-binding domains (Sander et al., 2011) with the VP16 activation domain (Figure 3B) or a KRAB repression domain (Figure 3A), as shown on Figure 2. To asses the activity of designed TAL regulators, we also designed reporter plasmids, which contain several repeats of TAL binding sites upstream of either a CMV promoter (repression) or a minimal promoter (activation) (Figure 3C).

In addition to the synthesis of new TAL effector-based parts and their characterization, our team also improved a part which was deposited in the Registry by the Slovenian iGEM2010 team. They synthesized a TAL effector, named NicTAL, which did not work as expected in mammalian cells. We discovered that a subdomain next to the DNA-binding domain was missing, because the requirements for the functional TAL binding domains have not been known two years ago. We linked the missing subdomain to the DNA-binding domain of NicTAL from the Registry. Additionally we prepared chimeric proteins of the NicTAL-DNA binding domain and KRAB or VP16 domains, generating another repressor and activator pair and demonstrated the newly acquired functionality of the NicTAL-based regulators.

Designed repressors

We designed and tested three different variants of TAL DNA-binding domain fusions with the KRAB repression domain. KRAB was placed either on both termini or on the N- or C-terminus of the TAL DNA-binding domain (Figure 3A). All tested constructs with the four different TAL domains exhibited over 90% repression of the reporter plasmid (with the exception of KRAB:TALD). We expected to observe a difference in repression due to potential clustering of KRAB-binding proteins, but no significant variation between constructs was noticeable. Our conclusion is that the position of the effector (regulator) domain does not influence the binding and repression ability of the designed TAL repressors. All further experiments were performed with TAL:KRAB fusions.

Figure 4. Schematic representation of repression experiments. (A) In the absence of a TAL repressor, the reporter gene is constitutively expressed. (B) When a TAL repressor is expressed, it binds to its respective DNA-binding site upstream of the CMV promoter and represses transcription of the reporter gene through KRAB domain-mediated transcriptional silencing.
Figure 5. TAL repressors strongly inhibit expression of reporter genes. HEK293T cells were cotransfected with TAL repressors under the control of a CMV promoter (50 ng), and with a firefly luciferase reporter plasmid (Figure 3C) containing 10 DNA-binding sites for the designated TAL repressor upstream the CMV promoter (10 ng). Along with the tested constructs we transfected cells with 5 ng of Renilla luciferase under the HSV-TK promoter as transfection control. Luciferase activity was measured 3 days post-transfection. All experiments were executed in 3 biological replicates and repeated more than 3 times with similar results.

Results show that the previously non-functional NicTAL2010:KRAB fusion (NicTAL DNA-binding domain constructed by the iGEM2010 team Slovenia) acquired functionality by the N-terminal addition of a subdomain to the TAL DNA-binding domain (Figure 5). An excellent repression ability of NicTAL2012:KRAB (the improved version by the 2012 team) was observed. The NicTAL2012 repressor was further characterised by testing the effect of a different number of binding sites upstream of the PCMV promoter on the inhibition of reporter expression. Results presented in Figure 6 show that the maximal effect of the bound TAL regulator plateaus at 7 or more copies of binding sites per operator. In all further experiments we used plasmids with 10 copies of TAL DNA-binding sites.

Figure 6. Number of DNA-binding sites specific for NicTAL12:KRAB repressor dictates the efficiency of inhibition of reporter expression. HEK293T cells were cotransfected with NicTAL repressors under CMV promoter (50 ng), and firefly luciferase reporter plasmids (Figure 3C) with different number of NicTAL binding sites upstream of the CMV promoter (100 ng). Luciferase activity was measured 3 days post-transfection. The experiment wes executed in 3 biological replicates and repeated over 3 times with similar results.

Designed activators

Figure 7. Schematic representation of activation experiments. (A) In the absence of a TAL activator, there is no expression of the reporter gene. (B) When TAL activator is present, it binds to its DNA-binding site upstream of the minimal promoter and activates transcription of the reporter gene.

Due to the mechanism of action of the VP16 domain and our previous results with the TAL:KRAB repressor (Figure 5, the position of the effector domain does not influence repression), we decided to test only the C-terminal variant of the TAL:VP16 fusion (Figure 3A). Both tested TAL activators exhibited over 1500-fold activation of the mCitrine reporter at reporter to activator ratios 1:2. In addition, we have confirmed that the minimal promoter used to drive the expression of the reporter gene shows no (or minimal) leakiness - this trait makes this promoter an excellent element for genetic systems, where tight transcriptional regulation is needed.


Deng, D., Yan, C., Pan, X., Mahfouz, M., Wang, J., Zhu J. K., Shi, Y., and Yan, N. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 335, 720-723.

Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T.Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 40, 7584-95.

Mak, A. N., Bradley, P., Cernadas, R. A., Bogdanove, A. J., and Stoddard, B. L. ( 2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335, 716-719.

Miller, J. C , Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., Meng, X., Paschon, D. E., Leung, E., Hinkley, S. J., Dulay, G. P., Hua, K. L., Ankoudinova, I., Cost, G. J., Urnov, F. D., Zhang, H. S., Holmes, M. C., Zhang, L., Gregory, P. D., and Rebar, E. J. (2011) A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143-148.

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29, 697–698.

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

Zhang, F., Cong, L., Lodato, S., Kosuri, S., Church, G. M., and Arlotta, P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149-153.

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