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 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 ∞

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.

1.RFP from linear RNA (with stop codon)
2.RFP from circular RNA (with stop codon)
3.RFP from circular RNA (without stop codon)
4.RFP from circular RNA (with the stop codon of mRNA circular device)
S.supernatant
P.precipitation

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)
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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1000
  Illegal AgeI site found at 1112
  
• 1000
  COMPATIBLE WITH RFC[1000]