The laboratory of Barry L. Stoddard, Ph.D., Fred Hutchinson Cancer Research Center

http://2012.igem.org/Team:Freiburg

GATE KIT FOR EASY GENOME ENGINEERING

Let us tell you a fabulous TALE Aim of our project is providing future iGEM students with a ready-to-use toolkit for fast and easy assembly of TAL Effectors (TALEs). From the sequence of your gene to a custom TALE in just one reaction.

The future of TALE

11111 nuclease

Transcription -factor

1. 2. 3. 4. 5. 6.

AA

AT

AG

CG

GG

A T G C

Effector protein

Legend:

Target Sequence: T-AATGAGGCATCT-T

+ + + + + +

1.AA + 2.TG + 3. AG + 4. GC + 5. AT + 6.CT + TF (VP64)

TAL effectors Transactivator-Like Effectors (TALEs) currently revolutionize the way researchers manipulate DNA with exceptional site specificity1. Originally derived from Xanthomonas spp., this type of protein comprises an effector domain and a modular DNA binding domain that can be rationally designed to bind to virtually any target sequence of DNA.

Over the past two years, universal endonucleases (TALENs) and transcription factors (TAL-TFs) have been tested in various organisms ranging from bacteria to humans.

Figure 1: In this picture, you can see a TAL protein bound to its target DNA sequence. The different colors indicate the different repeats, each of which specifically interacts with one base in the target sequence.

According to most existing protocols2, TALE assembly requires several weeks of work and substantial lab skills. In order to bring this technology within reach for iGEM students, we invented an extremely fast and easy TALE assembly strategy and developed a TALE platform with expression plasmids and new classes of TALEs. With our so-called Golden gate cloning-based Automatable TAL Effector (GATE) assembly kit, future iGEM students will be able to precisely manipulate genomic loci easier and faster than anyone else in the world. Golden Gate Cloning is a cloning technique based on the function of type IIs restriction enzymes like BsmB1. These enzymes recognize a certain site, like every restriction enzyme, but cut slightly downstream of this site. This makes it possible to create multiple sticky ends with one enzyme in one reaction and therefore assemble multiple parts in a specific order in one reaction. In our kit, we used direpeats instead of repeats, which cuts half the number of parts that need to be assembled in one step and thereby enables us to built a whole TALE in a single reaction.

Open the GATE

With our kit, we transform a “high tech” to a “high school” application, bringing TALE production within reach for every researcher. Furthermore, the GATE assembly kit is not only simple to use for researchers but also automatable - as we could demonstrate by implementing our GATE assembly kit into a pipeting robot. Moreover, with our so-called Plug-and-Play Effector Cassette (PPEC), we enable future iGEM students to fuse any enzyme to the TAL protein and create their own classes of TAL effectors.

Resources:

To build your custom TAL effector, pipet the corresponding six direpeats into a PCR tube. In the next step, add a backbone, containing for example VP64 for transcriptional activation, BsmB1 and T4 ligase and put the tube into your thermocycler. After a 2.5 hour run, your TAL plasmid is ready for transformation. For a complete step by step manual including thermocycler programs as well as the complete list of compounds for the Golden-Gate reaction, please visit our iGEM Wiki page.

Target Sequence: AATGAGGCATCT

+ + + + + +

1.AA + 2.TG + 3. AG + 4. GC + 5. AT + 6.CT + TF (VP64)

Combine the sequences in one step of Golden Gate Cloning to get the ready-to-use sequence of your entire TAL-TF effector.

The VP64 domain will activate the transcription of the GOI

T T A C T C C G T A G A N N N N N N N N N N N

A A T G A G G C A T C T N N Gene of Interest Promotor

Figure3: Assembly of a TAL-Transcription factor fusion protein. After transfection of the TALE plasmid, the protein binds to the targeted sequence either on another plasmid or directly in the host genome. In the case of transcription factor fusion, this leads to activation of the downstream promoter and therefore to the enhanced transcription of the targeted gene.

Results and Testing In order to test our GATE assembly kit, we used a vector containing a secreted alkaline phosphatase (SEAP) under the control of a minimal promoter. We targeted the latter with a TAL effector that was coupled to a VP64 transcription factor.

1

SEAP +

Measureable at 405 nm in a spectrophotometer

para-nitrophenylphosphat para-nitrophenol

We could demonstrate, that we are able to enhance the transcription of a SEAP gene under the control of a minimal promotor by targeting it with a TAL coupled to a VP64 domain. This shows the efficiency of specific transcriptional activation using a TAL effector.

Figure 3 (left), 4 (right) and 5 (below). Figure 5 illustrates the principle of a secreted alkaline phosphatase assay (SEAP assay). In figure 4, you can see the results of a TAL-transcription factor targeting the minimal promoter upstream of a SEAP gene. The enzymatic activity of SEAP is measured by the increase of absorption at 405 nm over a time of ten minutes. Figure 3 shows the average absorption after 9 minutes, including error bars and p-values. As expected, only the cotransfection of our SEAP plasmid and the TAL-TF plasmid showed strong SEAP expression.

Figure2: GATE assembly kit. To create the sequence of a 12 domain TALE protein, the 12 domains are splitted up into 6 double domains (DD). In our toolkit, we provide „intelligent“ DDs that already know their place in your finished protein. The only thing left to do is pick the right ones, put them in a thermocycler and wait for 2.5 hours.

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

Control TALTF SEAP ++

TAL-TF SEAP Assay at 9 min

p=0,00041

p=0,00043

p=0,00037

GATE

1. Bogdanove, A. J. & Voytas, D. F. TAL Effectors: Customizable Proteins for DNA Targeting. Science 333, 1843–1846 (2011).

2. Sanjana, N. E. et al. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7, 171–192 (2012).

Wyvekens, N., Siegel, D., Schneider, L., Böckelmann, L., Grassi, P., Dreßler, F., Stritt, F., Warmer, P., Scheller, L., Waehle, V., Herman, J., Grishin, D., Jerabek, L., Runge, K., Steitz, J., Fuchs, D., Küchlin, S., Knoll, N., Proksch, S.