Team:Kyoto/GSDD/Method

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Method

Description of the Mechanism and the Process to think them up

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First, to translate our thinking into reality, we thought about the following thing; for direct regulation, if there are any inner cellular substances or internal cellular chemical reactions, which change its composition or reaction rate or something like that, depending on the cycles of cell division. Then we put our eyes on “the End Replication Problem” found in the duplication of eukaryotic cell’s chromosomes. By repeating the process, both ends of each linear chromosome (telomeric sites) becomes shorter (about 1~200bps in yeast). So we thought whether we could take this mechanism into our design.

At the same time, we found a reference, by chance, in which a designed linear DNA was stabilized in E coli by the binding of lactose repressor to the both ends of designed repeated binding sequences. Also we found an online article about artificially designed chromosome for yeast, which is also stabilized and enabled to pretend like a natural chromosome in yeast (replicated by DNA polymerase and distributed into each daughter cell in each cell division). This chromosome is called “Yeast Artificial Chromosome” (YAC) and is used by many biologists in yeast gene cloning. YAC consists of three important parts; intermediate replication origin, both ends of telomeric sites, and centromeric site. From replication origin, chromosome replication starts at the time of chromosome replication. By telomeric sites, even if some end base pairs are cut off by the end replication problem, in YAC the chromosome can keep its ends length same by processing them with DNA repair mechanism. Centromere is essential site for exact chromosomal division. It can let the cell exactly distribute double chromosomes into each daughter cell.

Based on above knowledge, we designed two functionally different vectors; Timer vector and Bomb vector. Timer vector is linear DNA. We designed this to behave like YAC (this means it would behave in the same way with natural chromosome but has a big difference with YAC), and regulate a killing gene on Bomb vector. Bomb vector is circular DNA, and it consists of a killing gene regulated by a protein expressed from Timer vector. Then we determined to use yeast in our experiment.

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Timer vector has, at both ends, repeated sequences of LacI binding sequences, and has a replication origin and a centromeric site in its intermediate part. In addition, Timer vector has a constitutive LacI generator (which means constitutively active promoter and a yeast kozac sequence and LacI coding sequence) On the other hand, Bomb vector has a LacI regulated promoter and a killing gene with a yeast kozac sequence. We designed these two vectors to be put into the same cell. (Double transformation)

When yeast is transformed by these two vectors, we assume, the constitutively expressed LacI binds to the both ends of repeated sequences of Timer vector, and protects them from the degradation by exonuclease in cellular cytoplasm. At the same time, LacI binds to lac promoter also in Bomb vector, and it prevents the expression of killing gene. As a result, while Timer vector is protected from the attack by exonuclease and keep the constitutive expression of LacI, it can repress the killing gene on bomb vector. That is, while Timer exists and keeps LacI generator in its intermediate site, the cell can keep itself alive.

However as cell division repeats from the first double transformation, the both ends of Timer vector becomes shorter by the end replication problem, and after several divisions, the repeated sequences is completely lost, and then the Timer vector is degraded by exonuclease and the LacI generator is also lost. Finally, the cell express killing gene and kill itself.