Part:BBa_K1859024:Experience

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===Applications of BBa_K1859024===

We inserted this generator to E.coli and measure efficiency of circularization.
We extracted total RNA, reverse transcribed, conducted semi-quantitative PCR.
As a result, this generator circularized more efficiently than normal (no complementary sequence) one.[Gifu 2015 RESULT]

   The result of Semi-quantitative PCR was shown below. And, we made the graphs by using the raw data.

Fig.2 As a result of quantitative experiment of Circular mRNA

Table.1 The mean of the fluorescence　(tripartite)

Fig.3 Relationship of fluorescence and cycle number

From the left, normal, outside, insideⅠ and insideⅡ

Table.2 Ct value at fluorescence 1.90

  According to this result, we calculated Ct value which indicates 1.90 fluorescence intensity. These value is summarized right. Ct is a value that the more the gene template in PCR increases, the more Ct value decreases. If there is a difference between “C” and “D”, there is a difference in the quantity of template geneThus, the difference between “C” and “D” is small; the cyclization may be efficiency.As a result, Ct in " normal [BBa_1332011] " was 2.31, but “ outside [BBa_1859026] ”, “ inside Ⅰ [BBa_1859024]　 ”, “ inside Ⅱ [BBa_1859025] ” were 1.28, 2.02, 1.89 collectively. From these things, it can be said that the efficiency of cyclization rose with all devices which we designed in this time. Especially, in the case of “outside”, there was a difference with Ct more than 1 point than “normal”. When we think that a quantity of the gene doubles for 1 cycle in PCR simply, it can be said that the cyclic efficiency of “outside” was twice as high as that of “normal”.
  After all, it is thought that it fitted this splice site because it is a complementarity chain derived from the creature.

Table 3. Efficiency of circularization (relativity value)

	normal	outside	insideⅠ	insideⅡ
Efficiency of circularization (relativity value)	1.00	2.05	1.22	1.34

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