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.

Existence proof of circular mRNA

Summary　of the experiment

The existence of circular mRNA is confirmed by RNase processing. RNA is decomposed by RNaseA (endoribonuclease). Endogenous 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

	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.

cDNA solution 10ul

	Group1		Group2		Group3		Group4 (total RNA without 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	2.5 µL	2.5 µL	2.5 µL	2.5 µL	2.5 µL	2.5 µL	2.5 µL	2.5 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 liner(endogenous) mRNA)	2.5 µL		2.5 µL		2.5 µL		2.5 µL

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]