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]

Detecting circular mRNA
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.

Fig. As a result of qualitative experiment of Circular mRNA

1:normal(iGEM Gifu 2014) 2:outside complementarity 3:inside complementarityⅠ 4:inside complementarityⅡ

a…To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseR processing.

b…To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseR processing.

c…To detect the sequence of the circular mRNA in the cDNA derived from the non-treated RNA.

d…To detect the sequence of the linear mRNA in the cDNA derived from the non-treated RNA.

e…To detect the sequence of the circular mRNA in the non-treated RNA

f…To detect the sequence of the linear mRNA in the non-treated RNA

M…marker

According to the picture (show above), this generator secceeded in making the circular mRNA.

Semi-quantitative PCR

  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. We conducted reverse transcription PCR and PCR for the target. In the PCR, we performed 10 kinds of reaction which had the different number of cycles(12,14,16..30) for each sample. Then, we applied them in agarose gel and electrophoreses. And, we analyzed the data by using "imageJ".

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

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

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

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