Part:BBa_K1859026:Experience

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===Applications of BBa_K1859026===

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 existence of circular mRNA is confirmed by RNase processing. Endogenous RNA (linear RNA)(GAPDH) is decomposed by RNase R (exoribonuclease), but circular RNA is not decomposed.
  Double-stranded DNA from undecomposed RNA can be gained with RT-PCR. This is how the existence of circular mRNA is confirmed by the observation of the DNA with electrophoresis.

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

  We introduced plasmid of "normal [BBa_K1332011] ", "outside [BBa_K1859026] ", inside Ⅰ [BBa_K1859024] , and inside Ⅱ [BBa_K1859025] to E. coli, and pre-incubated each in 37℃ over night. Then we cultured with shaking the pre-culture liquids 100 µL with LB-Cm 10 mL in 37 for four hours and extracted total RNA from them with RNA extraction kit. After then, we reverse transcribed RNA by PCR and amplified certain region we targeted. One is a unique region of circular mRNA [ region C ], which includes joint region of circularization [ region D ]. The other is a common domain among circular and un-circular mRNA.

Fig. As a result of quantitative experiment of Circular mRNA

Table.1 The mean of the fluorescence　(tripartite)

Fig. 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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