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Team:Tsinghua-A
From 2014.igem.org
Contents |
Background
Transactivator-Like Effectors (TALEs) are a technology that once revolutionized the way researchers manipulate DNA with exceptional site specificity. TALEs are proteins secreted by Xanthomonas bacteria and they recognize DNA sequences through a central repeat domain consisting of a variable number of 34 amino acid repeats. There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence.
Assembly
The TALE assembly strategy uses the Golden Gate cloning method, which is based on the ability of type IIS enzymes to cleave outside of their recognition site. When type IIS recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs, termed fusion sites, to be ligated seamlessly. Since type IIS fusion sites can be designed to have different sequences, directional assembly of multiple DNA fragments is feasible. Using this strategy, DNA fragments can be assembled from undigested input plasmids in a one-pot reaction with high efficiency. We chose the native TALE AvrBs3 as a scaffold for customized assembly of TALE constructs. The central DNA binding domain of AvrBs3 is formed by 17.5 tandemly arranged 34 amino acid repeats, with the last half repeat showing similarity to only the first 20 amino acids of a full repeat. To reduce the risk of recombination events between the 17.5 highly homologous repeat sequences, we codon-optimized avrBs3 applying the codon usage. In a single Golden Gate cloning reaction, cloning efficiency is significantly reduced for assembly of 17 repeat modules. Therefore, we split the assembly in two successive steps. In the first cloning step, 10 repeats are assembled in one vector. The preassembly vectors confer SpecR and encode a lacZ-α fragment for blue/white selection. On both sides of the lacZ-α fragment a type IIS recognition sequence - BsaI - is positioned. Similarly, 11~17 repeats and NG-last-repeat are respectively ligated and inserted into another vector. After preassembly of the 10 and 7 and last repeats using BsaI, the intermediate blocks are released via Esp3I and cloned into the final assembly vector (modified pTAL1). Modified pTAL1 confers AmpR, and allows plasmid replication in E.coli. The vector pTAL1 also contains all elements of the final TALE expression construct, except the repeat modules.
Modeling
In modeling part, genetic algorithm is applied to TALE sequence optimization. We exploit amino acid degeneracy and alternate the nucleotides to reduce the repetition rate of DNA bases of the DNA sequence of TALE. Then the optimized TALE will be tested by biological experiments. We divide our algorithm into four parts. First, we alternate a random point of natural DNA sequence of TALE. It is the same as gene mutation. Thousands of mutational sequences form a population and we simulate the process of reproduction in computer. The population will be changed by exchange of parts of DNA. Then each sequence will be estimated by dynamic programming and given a score. The high scored sequences have higher possibility to survive and the lower ones are more likely to be obsoleted. After more than 200 generations, an optimized population will be created. And we choose a certain number of the sequences to be tested in the next biological test.
Report System
We construct a report system so as to test the reliability and efficiency of our ‘Telling TALE’. In this section, we test the TALE’s DNA binding ability and report it with a common report gene ‘RFP’. We attempt to put the target of TALE’s DNA binding target sequence inside the expression cassette of report gene and binding TALE can disrupt the express of report gene. We use iGEM standard parts to build our report system.
Reference
Weber E, Gruetzner R, Werner S, Engler C, Marillonnet S (2011) Assembly of Designer TAL Effectors by Golden Gate Cloning. PLoS ONE 6(5): e19722. doi:10.1371/journal.pone.0019722
