Difference between revisions of "Part:BBa K1332011"
| Line 130: | Line 130: | ||
<h3>SDS-PAGE</h3> | <h3>SDS-PAGE</h3> | ||
We confirmed the existence of the long-chain protein derived from the circular mRNA by SDS-PAGE. <br> | We confirmed the existence of the long-chain protein derived from the circular mRNA by SDS-PAGE. <br> | ||
| − | [[File:PROTEIN1.png]]<br><br>[[File:PROTEIN2.png]] | + | [[File:PROTEIN1.png|500px|]]<br><br>[[File:PROTEIN2.png|500px|]] |
There is a long-chain protein near a band that indicates 250 kDa. The molecular weight of a monomeric RFP is 25423.7(→ BBa_E1010), so we guess that the protein is not less than decameric RFP. | There is a long-chain protein near a band that indicates 250 kDa. The molecular weight of a monomeric RFP is 25423.7(→ BBa_E1010), so we guess that the protein is not less than decameric RFP. | ||
Revision as of 16:38, 10 October 2014
Histidine tag (8 AA) and RFP semi-permanent generator
This generator is capable of synthesizing a RFP (+histidine tag) polymer. This generator consists of a circularization device (5’ side), histidine tag (8 AA) and RFP (without stop codon) and a circularization device (3’ side). 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.
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 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
Protocol
1.RNase processing
Mix the following reagents.
| Group1(RNaseA) | Group2(RNaseR) | |
| RNaseA | 1 µL | |
| RNaseR | 1 µL | |
| buffer | 1 µL | |
| RNA solution | 9 µL | 8 µL |
| total | 10 µL | 10 µL |
Incubate them at 37 °C for 20 minutes.
2.RT-PCR
Mix the following reagents.
| Group1(RNaseA) | Group2(RNaseR) | Group3(non-treated) | |
| RNA solution(after RNase processing) | 8 µL | 8 µL | |
| RNA solution | 6 µL | ||
| pure water | 2 µL | ||
| Oligo(dT)15primer | 1 µL | 1 µL | 1 µL |
| Random primer | 1 µL | 1 µL | 1 µL |
| total | 10 µL | 10 µL | 10 µL |
Incubate them at 70 °C for 5 minutes.
Incubate them at 4 °C for 5 minutes.
Incubate them on ice.
Mix the following reagents.
| Nuclease Free Water | 4.58 µL |
| GoScript™ 5×reaction buffer | 12.2 µL |
| 25mM MgCl2 | 6.1 µL |
| 10mM PCR Nucleotide Mix | 3.05 µL |
| Recombinant RNasin Ribo nuclease Inhibitor | 1.52 µL |
| GoScript™ Reverse | 3.05 µL |
Add 10 µL of it each to group1,2,3.
Synthesize cDNA.
| Annealing | At 25 °C for 5 minutes |
| Elongation | At 42 °C for 60 minutes |
| Inactivation | At 70 °C for 15 minutes |
| At 4 °C for ∞ |
Mix the following reagents.
| Group1 | Group2 | Group3 | Group4 (total RNA without RNase processing and reverse transcription) | |||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
| Nuclease Free Water | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL |
| GoTaq 2×mix | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL |
| primer Fw (to detect circular mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| primer Rv (to detect circular mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| primer Fw (to detect linear(endogenous) mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| primer Rv (to detect linear(endogenous) mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| cDNA solution (Group1) | 10 µL | 10 µL | ||||||
| cDNA solution (Group2) | 10 µL | 10 µL | ||||||
| cDNA solution (Group3) | 10 µL | 10 µL | ||||||
| RNA solution (Group4) | 10 µL | 10 µL | ||||||
Do the PCR assay.
| At 94°C for 3 minutes | |
| At 94°C for 20 seconds | 35 cycles |
| At 58°C for 20 seconds | |
| At 72°C for 50 seconds | |
| At 72°C for 70 seconds | |
| At 4°C for ∞ |
3.Electrophoresis
Result
Positive: 3,5,6
Negative: 1,2,4,7,8
1. To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseA processing
2. To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseA processing
3. To detect the sequence of the circular mRNA in the cDNA derived from the RNA after RNaseR processing
4. To detect the sequence of the linear mRNA in the cDNA derived from the RNA after RNaseR processing
M. Marker
5. To detect the sequence of the circular mRNA in the cDNA derived from the non-treated RNA
6. To detect the sequence of the linear mRNA in the cDNA derived from the non-treated RNA
7. To detect the sequence of the circular mRNA in the non-treated RNA
8. To detect the sequence of the linear mRNA in the non-treated RNA
Data analysis
See the lane 7,8. → RNA is not detected by the electrophoresis, namely, the matter detected is cDNA.
See the lane 5,6,7,8. → The factor involved in the existence of cDNA is the ribonuclease processing.
See the lane 1,2,5,6. → The endoribonuclease decomposes the all RNA.
See the lane 3,4,5,6. → There is the RNA decomposed by the exoribonuclease.
Therefore, the RNA that is decomposed by the endoribonuclease but is not decomposed by the exoribonuclease exists. We think this RNA is the circular mRNA!
Proof of existence of long protein
SDS-PAGE
We confirmed the existence of the long-chain protein derived from the circular mRNA by SDS-PAGE.
There is a long-chain protein near a band that indicates 250 kDa. The molecular weight of a monomeric RFP is 25423.7(→ BBa_E1010), so we guess that the protein is not less than decameric RFP.
Western blotting
This experiment is now underway. The detail is in our wiki.
The ability of coloration
1.RFP from linear RNA (with stop codon)</br>
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 near, 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]