Difference between revisions of "Part:BBa K1332009"

Revision as of 03:14, 2 November 2014

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 (3'side)

Part which surrounded by red closing line is mRNA circularization device (3’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