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PROJECT
RESULT
EFFICIENCY
We introduced plasmid of 去年の、外側、内側① and 内側② 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 for PCR, in this sequence we targeted at domains. One is a unique domain of circular mRNA, which includes joint domain when circularization. Other is a common domain among circular and un-circular mRNA.
Picture : domain C and D
We conducted reverse transcription and PCR on this target.
In PCR, we prepared 10 kinds of reaction which had different number of cycles(12,14,16..30) for each sample and applied them in agarose gel and electrophoresed.
Describe of picture
We quantized fluorescence intensity(average of triplicate) and summarized them in terms of number of cycles and fluorescence intensity.
Table
Picture of 去年
Picture of 外側
Picture of 内側①
Picture of 内側②
According to this result, we calculated Ct value which indicates 1.9 fluorescence intensity.
Picture
Summary of the experiment
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. So the existence of circular mRNA is confirmed by the observation of the DNA with electrophoresis.
Flow of the experiment
Purpose: proving the existence of circular mRNA
Goal: finding the RNA that is decomposed by endoribonuclease but is not decomposed by exoribonuclease
Protocol:
1. RNase processing: to find the circular mRNA
2. RT-PCR: to synthesize cDNA and to detect the cDNA
3. synthesized from circular mRNA or endogenous RNA
Electrophoresis: to detect the DNA synthesized from the cDNA
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 2014 was 2.31, but “outside”, “inside①”, “inside②” 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 “2014”. 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 “2014”.
After all, it is thought that it fitted this splice site because it is a complementarity chain derived from the creature.
FUNCTION
We made 7 kinds of linker in this experiment.
We made parts which have these sequence of linker in the downstream of the 3’ side of the intron (Bba_K1332005) or the upstream of the 5’ side of the intron without stop codon (Bba_K1332003).
We constructed plasmids like following it.
In case of inserting these plasmid into E. coli, the following circular mRNA is expressed and the long chain protein is synthesized.
We inserted these plasmid into an E.coli and made it synthesize proteins and did SDS-PAGE using this proteins. If the protein is not boiled, we can do SDS-PAGE keeping it fluorescence because RFP’s structure is strong. Therefore we applied samples that were not boiled.
Fig.1 Fluorescence of protein after it was done electrophoresis.
Fig.2 Image of protein fixed and dyed after that
There was not fluorescence at the place of long chain protein. We found that long chain protein didn’t have a function.
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