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Revision as of 14:40, 18 September 2015
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PROJECT
RESULT
EFFICIENCY
According to this result, we calculated Ct value which indicates 1.9 fluorescence intensity.
RNase processing
The result of "RNase processing" was shown below.
According to the line e and f, RNA was not detected by the electrophoresis, namely, DNA did not contaminate.
According to the line b and d, linear mRNA was cleaved by exoribonuclease.
According to the line a and c, mRNA was not cleaved by exoribonuclease.
From the above, the circular mRNA existed in all samples.
Semi-quantitative PCR
The result of Semi-quantitative PCR was shown below [ raw data ]. And, we made the graphs by using the raw data.
Fig.
We compared each Ct value at the same 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" was 2.31, but “outside”, “inside ①”, “inside ②” 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.
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 before dying by CBB
Fig.2 after dying by CBB
There was not fluorescence at the place of long chain protein. We found that long chain protein didn’t have a function.
Reason of why BBa_K1859020 and BBa_K1859022 don’t synthesize the long chain protein
In this experiment, BBa_K1859020 and BBa_K1859022 didn’t generate the long chain protein. The reason of this phenomenon can be deduced in terms of the sequence of the circular mRNA.
We made prediction of synthesized mRNA’s secondary structure.
Picture of BBa_K1332011
Picture of BBa_K1859027
Picture of BBa_K1859028
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