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

Revision as of 15:15, 18 September 2015

PROJECT

EXPERIMENTS

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.

Fig1. the role of 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.

linker +		parts name
GGSGGS	3'side of the intron BBa_K1332005	BBa_K1859001
GSGSGS		BBa_K1859002
(GGSGGS)×2		BBa_K1859007
(GSGSGS)×2		BBa_K1859008
(GGSGGS)×3		BBa_K1859009
(GSGSGS)×3		BBa_K1859010
HHHHHH		BBa_K1859003

	+ linker	parts name
5'side of the intron BBa_K1332003	GGSGGS	BBa_K1859004
	GSGSGS	BBa_K1859005
	(GGSGGS)×2	BBa_K1859011
	(GSGSGS)×2	BBa_K1859012
	(GGSGGS)×3	BBa_K1859013
	(GSGSGS)×3	BBa_K1859014
	HHHHHH	BBa_K1859006

We made following generators by using these parts.

No.	X	Y	generator
A	—	—	R0010+ B0034+ K1332001+ B0015
B	BBa_K1332005	BBa_K1332003	BBa_K1332011
C	BBa_K1859001	BBa_K1859004	BBa_K1859027
D	BBa_K1859002	BBa_K1859005	BBa_K1859028
E	BBa_K1859007	BBa_K1859011	BBa_K1859020
F	BBa_K1859008	BBa_K1859012	BBa_K1859021
G	BBa_K1859009	BBa_K1859013	BBa_K1859022
H	BBa_K1859010	BBa_K1859014	BBa_K1859023
I	BBa_K1859010	BBa_K1859014	BBa_K1859023
J	BBa_K1859001	BBa_K1859006	BBa_K1859025
K	BBa_K1859003	BBa_K1859004	BBa_K1859024

   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.

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.

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)

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.

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".

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