Revision as of 19:28, 2 November 2014

mRNA circularization device (5� side)

This part consists of a promoter (lacI regulated)(BBa_R0010), The 3' side of the intron(+exon fragment) from td gene of T4 phage(BBa_K1332005) and RBS(BBa_B0034). The protein coding sequence that is inserted between this device and mRNA circularization device (3’ side) can be circularized. If you circularized the protein coding sequence (Its a stop codon have been removed.) with mRNA circularization device (3´ side) (endless translation)(BBa_K1332009), you can get a circular mRNA that is translated semi-permanently.

Circular Parts

Figure 1. mRNA circularization device (5'side)

A Part surrounded by red closing line is mRNA circularization device (5’side).(figure 1)

How to use

You need modifying the prefix and suffix of a protein coding sequence. There is a stop codon “TAG” in a restriction site in the prefix, so insert “AG” just before the stop codon to shift a reading frame. (figure 2)

Figure 2. Modifying the prefix of a protein coding sequence

There is a translation termination codon in the protein coding frame, so delete a part of the sequence to remove the translation termination codon. (figure 3)

Figure 3. Modifying the suffix of a protein coding sequence

And then after that, you insert the protein coding sequence between this device and mRNA cicularization device (3’ side)(endless translation). At last, you insert the plasmid into E.coli. (figure 4)
3' side device(endless translation) is K1332009

Figure 4. How to use Ciucular parts

Mechanism

After the plasmid was inserted into E.coli, it occurs reactions in vivo as follow. Through this mechanism, Circular mRNA is made and long-chain proteins are synthesized.(figure 5)

Figure 5. Mechanism of mRNA circularization

A circular mRNA consists of RBS, the protein coding sequence and 56bp fragments of the mRNA circularization device. (figure 6)

Figure 6. Fragments of the mRNA circularization device (Red"GT" is removed.)

Adjust the length of a circular mRNA to multiples of 3 to keep a pattern of the reading frame.

When protein coding is RFP coding, its results shows as follow.

Figure 7.In the case that RFP is inserted as protein coding.
An RFP which we use is BBa_K1332002. That's an RFP which combines with a Histidine tag.

The existence of the circular mRNA

ribonuclease processing

Summary of the experiment

The existence of circular mRNA is confirmed by ribonuclease(RNase) processing. We used two types of RNase. One is the endo-type RNase. This cleaves the RNA at random. The other is the exo-type RNase. This cleaves the RNA from end. In experiment, we prepared the linear mRNA(GAPDH) as a control. The linear mRNA is cleaved by either endo or exo-RNase. On the other hand, circular mRNA is cleaved by endo-RNase but not by exo-RNase. Because, circular mRNA has no end.

Figure 8.The difference between Linear RNA and circular RNA in two types of RNase(endo or exo) reaction
Double-stranded DNA derived from leaving RNA can be gained with reverse transcription(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 synthesized from circular mRNA or endogenous RNA
3. Electrophoresis: to detect the DNA synthesized from the cDNA

[http://2014.igem.org/Gifu/protocols2#CRD Go to the page of detailed protocol]

Result

Figure 9. Electrophoresis results

3.5.6 We detected band
1.2.4.7.8 We detected no band

there is a band in the circular mRNA fraction which used exo-RNase. This meanes that circular mRNA exists.

the sequence of Circular mRNA

summary of the experiment

To get the evidence of circularization, we determined the sequence of circular mRNA that contains joint by reverse transcription. The joint is made after the circularization.

Figure 10. Joint sequence = the evidence of circularization

Result

Figure 11. The evidence of circularization
This sequence is the same as we designed. This means that mRNA was circularized. And also, the sequence indicates that the reading frame cannot slip down if a ribosome rotates several laps.

Synthesis of long-chain proteins

Summary of the experiment

Confirm repeating translation by SDS-PAGE([http://2014.igem.org/Team:Gifu/Protocol#SDS protocol]).

Result

Figure 12. The result of SDS-PAGE

The proteins over 250 kDa were detected. This means that long-chain protein was synthesized by the circular mRNA that does not have stop codon.

Derived from RFP

Summary of the experiment

Perform the Western blotting using anti RFP antibody conjugated with peroxidase.

Result

Figure 13. The result of the Western blotting
The proteins over 250 kDa were bound with the antibody. It means that the long-chain protein derives from the RFP.

Circularize efficiency

Summary of the experiment

Reverse-transcribe the specific four fragments of DNA(A-D) and calculate the efficiency of　mRNA circularization by the MPN-PCR.

Result

The efficiency of circularization was 2.5%.
[http://2014.igem.org/Team:Gifu/Modeling Read more on the modeling.]

Quantitative determination of proteins

Summary of the experiment

Dye protein with the CBB and make the calibration curve between the strength of bands and the concentration of monomer RFP. Determine the quantity of the proteins.

Result

Figure 14. The strength of bands of monomer RFP
We calculated the sum of the stained area with the chromaticity from the picture. We made a calibration curve from “the sum of the stained area with the chromaticity of the gel” and “known concentration of the monomer solution”. The result that concentration of polymer and monomer is shown in the following table.

Table 1. The result that concentration of polymer and monomer
Concentration of the monomer RFP was 0.57(mg/mL), and the polymer RFP was 0.41(mg/mL).

discussion

ratio of existence about Circular mRNA and Linear mRNA and concentration of proteins in the same E. coli show as follow.
Figure 15. Ratio of existence about Circular mRNA and Linear mRNA and concentration of proteins
When the amount of Circular mRNA is about the same as Linear mRNA,

Figure 16. The difference of efficiency between Circular mRNA and Linear mRNA
Polymer RFP is 27 times as much weight as Monomer RFP.
Circular mRNA synthesized more proteins than Linear mRNA did.

The ability of coloration

Figure 17. The difference of coloration

The RFP (+histidine tag) polymer didn’t show the fluorescence.
Possible factor
1.The RFP polymer is too huge, so it becomes an inclusion body.
2.The repetitive amino acid sequences are too close, so the conformation of the RFP polymer is in disorder.

Sequence and Features