"
Team:Kyoto/GSDD/Method
From 2009.igem.org
Method
Description of the Mechanism and the Process to think them up
First, to translate our hope into reality, we thought about the following thing, if there are any inner cellular substances or internal cellular chemical reactions, which change its composition or reaction rate or anything like that, depending on the cycle of cell division. Then we focused on “the End Replication Problem” in duplication of eukaryotic cells chromosomes. By repeated the process, both ends of each linear chromosome (telomeric sites) becomes shorter (about 100~200bps in yeast). Then we wondered whether we could take this mechanism into our design.
At that time, by chance, we found a reference in which linear DNA, thrown into E coli, was stabilized by the binding of lactose repressor to the both ends of designed repeated binding sequences. At just the same time, we found an online article about artificially designed chromosome of yeast, which is 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 generally 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 duplication. By telomeric sites, after some ends length shorten by the end replication problem, YAC can keep its same ends length processed by DNA repair mechanism. Centromere is essential site for exact chromosomal division.
Based on these two knowledge, we designed two following functionally different vectors; Timer vector and Bomb vector.. Timer vector is linear DNA. It is supposed to behave like YAC (but has a big difference with YAC), and regulate killing gene on Bomb vector. Bomb vector is general circular DNA, and it consists of a killing gene regulated by a protein expressed from Timer vector. At this time, we determined to use yeast in our experiment (and, of course, optimized all gene design for yeast).
Timer vector has, at both ends, repeated sequences of LacI binding sequences, and has, in its intermediate site, replication originate site and centromeric site. And also Timer vector has a set of Lactose repressor constitutive 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 yeast kozac sequence and killing gene. We designed these two vectors to be put into yeast at the same time.
When yeast is transformed by these two vectors, we assume, the constitutively expressed LacI will bind to the both ends of Timer vector, and protect it from the degradation by exonuclease in cellular cytoplasm. And also LacI will bind to lac promoter in Bomb vector, and it will prevent the expression of killing gene. As a result, while Timer vector is protected from the attack by exonuclease and keep the 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 since the first transformation, the both ends of Timer vector becomes shorter by the end replication problem, and after several replication, the repeated sequences of LacI will be completely lost, and then the Timer vector will be degraded by exonuclease and the LacI generator will be also lost. Finally, the cell will express killing gene and kill itself.
