Part:BBa_K1332011
Histidine tag (8 AA) and RFP semi-permanent generator
This generator is capable of synthesizing a RFP (+histidine tag) polymer. This generator consists of the mRNA circularization device (5´ side)(BBa_K1332008), histidine tag (8 AA) and RFP (without stop codon))(BBa_K1332002) and mRNA circularization device (3´ side) (endless translation))(BBa_K1332009). A mRNA is circular, so translation continues semi-permanently. A synthesis of the RFP (+histidine tag) become possible by a simply transformation, but the coloration of RFP is weak.
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). 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.
When protein coding is RFP coding, its results shows as follow.
Figure 7.
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 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.
Result
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
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
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
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.
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.
When the amount of Circular mRNA is about the same as 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
1.RFP from linear RNA (with stop codon)
2.RFP from circular RNA (with stop codon)
3.RFP from circular RNA (without stop codon)：using this device
4.RFP from circular RNA (with the stop codon of mRNA circular device)
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1000
Illegal AgeI site found at 1112 - 1000COMPATIBLE WITH RFC[1000]
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