Team:Kyoto/GSDD/Results

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Experimental Reslts

Result-1 Microgene Polymerization Reaction (MPR) for constructing repetitive LacI binding sites

We achieved constructing repetitive sequence of LacI binding site. A certain length of LacI binding site is required in both ends of Timer vector. We expected that LacI binds to this site and protects the both ends of Timer vector from the degradation caused by exonuclease, so the both ends of Timer vector become shorter per duplication by the end replication problem. In short, the repetitive sequence counts the time. As a result, we constructed a set of repetitive sequence that contains multiple LacI binding site by MPR (Microgene Polymerization Reaction).

It was suggested that the temperature during MPR influences the length of the acquired sequence. Because we aimed to construct a set of repetitive DNA with different lengths, we investigated and optimized the temperature condition during MPR. After getting repetitive sequences, we inserted them into plasmid by flush end ligation, and designed the part that has repetitive sequence.

1)Experimental conditions in MPR

We tried various conditions in the temperature X. The result indicated that the temperature during MPR influences the length of the acquired sequence. At last, we programmed 72 o C of the temperature X. Fig shows the repetitive sequence that we used for designing parts. As shown in Fig. 4, we succeeded in constructing the polymerized microgenes with different number of LacI binding sites.

2)Designing the repetitive part of LacI binding site

We can not embed restriction site in the both ends of repetitive sequence part because it is consists of repeating short DNA. Embedding restriction site in each short primer leads to the degradation into small parts when digesting by enzyme. Therefore, we inserted it into plasmid by blunt end ligation. The repetitive part was inserted into PSB1A2 which has blunt end. In this process, we phosphorylated PSB1A2, and dephosphorylated the repetetitive. We purified them before ligation. In conclusion, we successfully designed and constructed the repetitive sequences with the multiple protein binding sites by MPR. This is the simple method to construct any repetitive DNA sequence. Thus, it might be applicable to synthesize other repetitive sequences with different lengths in a tailor-made manner.

Result-2 Construction of Timer vector that contains LacI generator and TIMER.

ここにTIMERベクタのコンストラクションスキームなど入れる

Result-3 Construction of the constitutive LacI expression vector

同様

Result-4 Analysis of the constructed TIMER vector in yeast

同様

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

If we model Timer vector, we can upgrade the timer function, and apply Timer vector to more various uses. In order to model it, it is necessary to evaluate the time to that the repetitive sequence is completely lost and Timer vector is degraded. We need to clear the length of DNA which becomes short.

The both ends of Timer vector become short because of the end replication problem as already discussed. However, the both ends are also degraded by exonuclease because the binding LacI don’t completely protect and stabilize them. Therefore, evaluation for the inhibition of degradation is important.

In order to evaluate the inhibition by binding LacI, we need to clear the length of linear DNA which becomes short not by the end replication problem but by exonuclease, after the Timer Vector is transformed. In previous work, they tried to evaluate the inhibition of degradation, binding LacI in ends. They quantify the inhibition by the DNA expression level, the amount of fluorescence of GFP. They compared (a) and (b) below. They observed them in RTS (rapid translation system).

(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 amount of fluorescence in (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 in ends inhibit the degradation by exonuclease. In previous work, they quantified the inhibition not by the length of degraded DNA but by the expression level, 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. We thought of two following ideas to clear them.

The first idea is to observe linear DNA which have various length of LacI binding site in both ends, and measure the time to stop the expression of GFP. In this idea, 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. So the second idea is supposed to be more realistic. It is to 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. This is expected to be possible by some experiments noted above.

The instability aspect of linear vector

The ends of DNA double-strand break are repaired by homologous recombination or nonhomologous end-joining after they are degraded by exonuclease inside the cell. Therefore, linear vector is supposed to be instable because there is a possibility of recombination with other DNA or circularization in addition to DNA degradation. Binding LacI or other DNA binding protein in both ends is expected to inhibit degradation, recombination and circularization. For example, telomeric DNA inside the cell has such a mechanism.

Timer vector has LacI in both ends. There still may be the instability aspect of it. In order to reinforce this end protection, the following ideas are likely to be effective.

1. Use other binding protein which has stronger binding force, instead of LacI.

1. Use mutant yeast with loss of function of a protein related DNA degradation.

By adopting these ideas, suppression of the instability is supposed to be stronger.