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Team:Freiburg/Project/Overview
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| - | TALEs make sequence-specific genome modification much easier that before and therefore attracts great interest in the synbio research community and beyond. Interestingly, many of the researchers who hold the patents on TALEs also released open source toolkits for TALE assembly for academic research. However, most strategies of TALE gene assembly published thus far rely on a hierarchical procedure, that is very time consuming, laborious and not automatable. | + | <div align="justify">TALEs make sequence-specific genome modification much easier that before and therefore attracts great interest in the synbio research community and beyond. Interestingly, many of the researchers who hold the patents on TALEs also released open source toolkits for TALE assembly for academic research. However, most strategies of TALE gene assembly published thus far rely on a hierarchical procedure, that is very time consuming, laborious and not automatable. |
Therefore we herein describe the Golden Gate cloning-based TAL Effector (GATE) Assembly platform, which enables literally everyone to produce low-cost, tailored TALEs within a few minutes of labwork and basic lab equipment. Moreover, we have automated this strategy and produced different TAL Effector Transcription Factors with 96 % success rate faster than any other method published before. | Therefore we herein describe the Golden Gate cloning-based TAL Effector (GATE) Assembly platform, which enables literally everyone to produce low-cost, tailored TALEs within a few minutes of labwork and basic lab equipment. Moreover, we have automated this strategy and produced different TAL Effector Transcription Factors with 96 % success rate faster than any other method published before. | ||
Revision as of 21:55, 26 September 2012
The GATE Assembly Kit
Therefore we herein describe the Golden Gate cloning-based TAL Effector (GATE) Assembly platform, which enables literally everyone to produce low-cost, tailored TALEs within a few minutes of labwork and basic lab equipment. Moreover, we have automated this strategy and produced different TAL Effector Transcription Factors with 96 % success rate faster than any other method published before.
Review of existing TALE construction methods
The 96 direpeats
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We took the sequences of the four already known TAL repeats A,C,G,T and combined them into 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 integrated into six versions marked with different terminal sequences, one version for every place of our final six direpeat TAL protein. Because we didn't want to buy six times 16 different synthesised direpeats we came up with a plan to produce them by ourselves. We created six primer pairs, each primer with a common part matching all of the direpeats and an unique overhang contacting the direpeat it binds to.
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Because we did not feel comfortable requiring six different restriction enzymes 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. This way it was possible for us to create different sticky ends with just one enzyme. Therefore we are able to ligate six different parts in the right order in one PCR step.
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After finshing these 96 different extension PCR's we had to ligate the products into the orignial iGEM BioBrick vector and finally got our full library of 96 unique direpeats.
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