Part:BBa_K1332009
mRNA circularization device (3 side) (endless translation)
This part consists of the The 5´ side of the intron(+exon fragment) from td gene of T4 phage without stop codon(BBa_K1332003) and a double terminator(BBa_B0015). The protein coding sequence that is inserted between this device and mRNA circularization device (5’ side)(BBa_K1332008) can be circularized. If you circularized the protein coding sequence (Its stop codon have been removed.), you can get a circular mRNA that is translated semi-permanently.
Circular Parts
Figure 1. mRNA circularization device (5'side)
Part which 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). At last, you insert the plasmid into E.coli. (figure 4)
(3' side device 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.
The existence of the circular mRNA
Summary of the experiment
The existence of circular mRNA is confirmed by RNase processing. RNA is decomposed by RNaseA (endoribonuclease). Endogenous RNA (linear RNA)(GAPDH) is decomposed by RNaseR (exoribonuclease), but circular RNA is not decomposed. Double-stranded DNA derived from undecomposed RNA can be gained with 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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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