Part:BBa_K1332008
mRNA circularization device (5 side)
This part consists of a promoter (lacI regulated), the 3´ side of the intron in td gene of T4 phage and RBS. 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) for endless translation, 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
A circular mRNA consists of RBS, the protein coding sequence and 56bp fragments of the mRNA circularization device. (figure 5)
Figure 5. 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
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.
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
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.
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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