Difference between revisions of "Team:Gifu/Experiment-page"

   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.

   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.

   We used BBa_K1859026 for generator of outside complementarity.

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.

image of BBa_K1859024(inside complementarityⅠ)


image of BBa_K1859025(inside complementarityⅠ)

RNase processing

  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 ① [BBa_K1859024] , and inside ② [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 : region C and D

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".