"

From 2012.igem.org

Home	Team	Official Team Profile	Project	Parts Submitted to the Registry	Modeling	Notebook	Safety	Attributions

																					Building a Toolkit	Using the Toolkit		Experiments

Building a Toolkit

Part 1: The 96 direpeats

In this first part we explain the theory of our project and the way we took creating our TAL toolkit. You will understand why our toolkit looks like it looks and whats the theory behind its mechanisms. If you look for a tutorial on how to use the toolkit look at the "using the toolkit" section in the project part.

We took the sequences of the four already known TAL Repeats A,C,G,T and combined them to 16 new, so called direpeat sequences. These 16 direpeats were ordered as gene synthesis products.
Now the real work began, to start building TAL Proteins we needed to expand our 16 direpeats a second time. Each of the 16 direpeats needed to be split into six versions, one version for every place of our final six direpeat respectevly twelve monorepeat TAL Protein. Because we didnt wanted to buy six times 16 different synthesised direpeats we came up with a plan to produce them by ourself. We created six primer pairs each primer with a common part matching everyone of the direpeats and an unique overhang to extend the direpeat it binds to.

Because we didnt intend to require six different restriction enzyms in the final pcr to produce a twelve direpeat TAL we used a technic called golden gate cloning. The technic uses the type two restriction enzyme BsmB1 and its ability to cut DNA slightly downstream of the recognition site. With this it is possible for us to create different sticky ends with using just one enzyme. Therefore we are able to ligate six different parts in the right order in one pcr step.

After finshing these 96 different extension pcr's we had to ligate the products into the orignial igem biobrick vektor and finally got our full library of 96 unique direpeats

Part 2: The TAL vektor

The next step of our project was the creation of a working vektor in which we could ligate our TAL protein for tranformation or transfection.