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Team:Kyoto/GSDD/Results
From 2009.igem.org
Reslts & Discussion
The difference between our designed device and YAC
In the case of YAC, both ends is teromeric site so when some end sequences of them cut off by the end replication problem, then the cut sequences will repaired and teromere keep its sequence. But in the case of our parts, the cut sequence will not repaired and the length will become shorter as cell division repeated. But the most problem is the cut length is different with each cell. We assumed the cut length of yeast in each one cell division might be about 100-200 base pair, this length is same with the average primer sequence in the ragging chain replication of yeast cells.
Evaluation for the inhibition of degradation by binding LacI
The both ends of Timer Vector become short because of end replication problem as already discussed. However, the both ends are also degraded by exonuclease because the binding LacI don’t protect and stabilize them completely.
It is necessary to evaluate the inhibition of degradation by binding LacI if we model Timer Vector. This model enables us to apply our Timer Vector to various uses. In order to evaluate the inhibition of degradation by binding LacI, we need to clear the length of linear DNA which is degraded not by the end replication problem but by exonuclease itself, after the Timer Vector is transformed.
In previous work, they tried to evaluate the inhibition of decomposition by binding LacI. They quantify the inhibition by the amount of observed fluorescence of GFP. They compared (a) and (b) below. They observed them in RTS ( ?? 大腸菌無細胞発現系の略称 ).
(a) linear DNA which can express GFP
(b) linear DNA which can express GFP and have LacI binding site in both ends
After (a) and (b) were expressed, the fluorescence amount of (b) was more than (a). In addition, when they increased the concentration of LacI in RTS, the difference of them became bigger. This work indicated that LacI inhibit the degradation by exonuclease. In previous work, they quantified the inhibition not by the length of degraded DNA but by the expression, and they used E coli. No studies have ever clear the length of DNA degraded by exonuclease and the length protected by LacI in Saccharomyces cerevisiae.
There are two possible ways to measure the length.
(1) Observe linear DNA which have various length of LacI binding site in both ends, and measure the time to stop the expression of GFP Much data is necessary to estimate the parameters to model it. It is thought to be difficult to estimate the parameters, because this data is supposed to vary because the end replication problem itself doesn’t happen evenly.
(2) Stop the duplication, and measure the length of linear DNA which is shortened only by exonuclease
After the Timer Vector is transformed, we stop the duplication in some way. We can observe the expression of GFP, and the time to stop the expression. This enables us to quantify the inhibition by the length of degraded DNA, where the end replication problem doesn’t occur.
To sum up, in order to model the Timer Vector, we need to evaluate the length of DNA degraded by exonuclease besides the end replication problem. We set up two possible ways to measure the length.
