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PROJECT
EXPERIMENTS
Developing the efficiency of circularization
In last year, we made a circular part by cloning splicing site from an intron of T4 phage. However, the efficiency of circularization was only 2.5%. Therefore, we wanted to circularize mRNA efficiently this year.
About the complementary sequences at outside of both splicing sites
There are the complementary sequences inside the splicing site in this intron. We thought that the sequence brings one splicing site close to the other one and ensures the reaction takes place.
Therefore, we cloned splicing sites with complementary sequences in intron and made it into parts. Moreover, we made the device which express circular mRNA by using this part, and made its express in E. coli. Then, complementary sequences are included in outside of both splicing sites in RNA.
Fig2. Replacing the splicing site and parts constructed
We used BBa_K1859026 for generator of outside complementarity.
Fig3. image of BBa_K1859026
About the complementary sequences at inside of both splicing sites
In case of designing complementary sequences at inside of both splicing sites, its sequence need including in a circular mRNA expressed in E. coli. We wanted to synthesize long chain proteins by making a circular mRNA translated semi-permanent. We had to design a complementary sequence which codes functional protein because the complementary sequence for inside was translated.
We used BBa_K1859024(inside complementarityⅠ) and BBa_K1859025( inside complementarityⅡ) for generator of inside complementarity.
Fig4. image of BBa_K1859024(inside complementarityⅠ)
Fig5. image of BBa_K1859025(inside complementarityⅠ)
GGSGGS and HHHHHH sequence which they are likely to be linker are adopted for complementary sequence at inside circular mRNA, for these two are complementary each other.
 Fig6. Inside complementary Ⅰ and Ⅱ
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.
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
4. Electrophoresis: to detect the DNA synthesized from the cDNA
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".
Making functional long chain protein
In our last study, our long chain protein lost its function. As a reason, we had an idea that the protein lost its holding structure because it was much longer. Therefore, we aimed to synthesize functional long chain proteins by designing some linkers in this year.
Fig13. the role of the linker
The requirements for amino acids that can be good linkers are less of steric effects and strong in hydropathy. Glycine and serine fulfill this requirements. Glycine’ side chain has almost no steric effects. On the other hand, serine has some steric effects but it has powerful hydropathy because of its hydroxyl side chain. According to Mr. Yabe, because histidine has quite strong hydropathy, though it has big steric effects, it is
possible that histidine can work as a linker.
Then, we designed some linkers [ see right ]. "GGSGGS", "GSGSGS", and "HHHHHH" were designed as linkers. We combined these sequence and inserted. The pattern of combination was shown below.
The 3'side of the intron
[BBa_K1332005]
conjuncted linker to keep folding of proteins.
Also,the 5'side of the intron
[BBa_K1332003]
conjuncted linker to keep folding of proteins.
Fig15. Combination of linker and 3’intron[left] / Combination of linker and 5’intron[right]
Table2. linker parts of 3’side [left] / linker parts of 5’side [right]
We made following generators by using these parts.
Fig16. Outline of the plasmid we constructed
Table3. Parts introduced into X, Y and provided generator
 Fig17. Localization of linker sequence after self-splicing
Amino acid chains synthesized from circular mRNA are constructed from repetition in amino acid of target protein, amino acid from ribozyme and amino acid from RBS. In this experiment, we inserted linker sequences into inside of both splicing sites. Therefore, protein chains synthesized from circular messenger RNA that include linker sequences repeat linker sequence, amino acid from RBS, target protein, linker sequence and amino acid from ribozyme.
Generally speaking, length of linker is required of one-forth of diameter of target protein. In the case of RFP, that condition is surely cleared by six amino acid, but it is unknown which linkers are appropriate in order to synthesize poly RFP protein. Therefore, we examined some types of amino acids and length of linker and choice the best linker in this experiment.
We inserted these plasmid into an E. coli and made it synthesize proteins and performed SDS-PAGE by using these proteins. We did SDS-PAGE without boiling to check fluorescence of RFP. Because RFP’s structure is strong, we can see the fluorescence.
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