Device

Part:BBa_K1332008

Designed by: Kenta Nomura   Group: iGEM14_Gifu   (2014-09-26)
Revision as of 22:33, 1 November 2014 by Fukufuku (Talk | contribs)

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

Cirpart1.png
Figure 1. mRNA circularization device (5'side)

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

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 1)
Prefix.png
Figure 1. 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 2)
Suffix.png
Figure 2. Modifying the suffix of a protein coding sequence

Howtouse1.png



A circular mRNA consists of RBS, the protein coding sequence and 56bp fragments of the mRNA circularization device. (figure 3)
Cyc-seq.png
Figure 3. 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 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 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.
Gifupartreg1.png 

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None