Difference between revisions of "Part:BBa K1332011"
 
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<partinfo>BBa_K1332011 short</partinfo>
 
<partinfo>BBa_K1332011 short</partinfo>
  
−
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.
+
This generator is capable of synthesizing a RFP (+histidine tag) polymer. This generator consists of the mRNA circularization device (5´ side)([https://parts.igem.org/Part:BBa_K1332008 BBa_K1332008]), histidine tag (8 AA) and RFP (without stop codon))([https://parts.igem.org/Part:BBa_K1332002 BBa_K1332002]) and mRNA circularization device (3´ side) (endless translation))([https://parts.igem.org/Part:BBa_K1332009 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.
  
−
<h3>Existence proof of circular mRNA</h3>
+
 
−
<h4>Summary　of the experiment</h4>
+
 
−
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.
+
[[File:Gifu RFP generator.png|500px|]]<br>
 +
<b>Figure 1.In the case that RFP is inserted as protein coding. </b><br>
 +
<b>Link:</b>
 +
<ul>
 +
<li>An RFP which we use is [https://parts.igem.org/Part:BBa_K1332002 BBa_K1332002].
 +
That's an RFP which combines with a Histidine tag.</li>
 +
<li>[https://parts.igem.org/Part:BBa_K1332011 BBa_K1332011]:Histidine tag (8 AA) and RFP semi-permanent generator</li>
 +
</ul>
 +
 
 +
<h2>The existence of the circular mRNA</h2>
 +
<h3>ribonuclease processing</h3>
 +
<h4>Summary of the experiment</h4>
 +
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.
 
<br>
 
<br>
 +
[[File:gifupartreg1.png|500px|]]
 +
<br>
 +
<b>Figure 2.The difference between Linear RNA and circular RNA in two types of RNase(endo or exo) reaction  </b>
 +
<br>
 +
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.
 +
<br>
 +
 +
 +
 
<h4>Flow of the experiment</h4>
 
<h4>Flow of the experiment</h4>
 
Purpose: proving the existence of circular mRNA<br>
 
Purpose: proving the existence of circular mRNA<br>
 
Goal: finding the RNA that is decomposed by endoribonuclease but is not decomposed by exoribonuclease.<br>
 
Goal: finding the RNA that is decomposed by endoribonuclease but is not decomposed by exoribonuclease.<br>
 
Protocol: <br>
 
Protocol: <br>
−
1. RNase processing: to find the circular mRNA<br>
+
1. RNase processing: to find the circular mRNA<br>
−
2. RT-PCR: to synthesize cDNA and to detect the cDNA synthesized from circular mRNA or endogenous RNA<br>
+
2. RT-PCR: to synthesize cDNA and to detect the cDNA synthesized from circular mRNA or endogenous RNA<br>
−
3. Electrophoresis: to detect the DNA synthesized from the cDNA<br>
+
3. Electrophoresis: to detect the DNA synthesized from the cDNA<br>
 
<br>
 
<br>
−
<h4>Protocol</h4>
+
[http://2014.igem.org/Gifu/protocols2#CRD Go to the page of detailed protocol]
−
1.RNase processing<br>
+
−
<table class="table" border="2">
+
−
<tr><td></td><td>Group1(RNaseA)</td><td>Group2(RNaseR)</td></tr>
+
−
<tr><td>RNaseA</td><td>1 &micro;L</td><td></td></tr>
+
−
<tr><td>RNaseR</td><td></td><td>1 &micro;L</td></tr>
+
−
<tr><td>buffer</td><td></td><td>1 &micro;L</td></tr>
+
−
<tr><td>RNA solution</td><td>9 &micro;L</td><td>8 &micro;L</td></tr>
+
−
<tr><td>total</td><td>10 &micro;L</td><td>10 &micro;L</td></tr>
+
−
</table>
+
 
<br>
 
<br>
−
Incubate them at 37 &deg;C for 20 minutes.<br>
 
 
<br>
 
<br>
−
2.RT-PCR<br>
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<h4>Result</h4>
−
Mix the following reagents.<br>
+
[[File:gifupartreg2.png|500px|]]<br>
−
<table class="table" border="2">
+
<b>Figure 3. Electrophoresis results</b>
−
<tr><td></td><td>Group1(RNaseA)</td><td>Group2(RNaseR)</td><td>Group3(non-treated)</td></tr>
+
 
−
<tr><td>RNA solution(after RNase processing)</td><td>8 &micro;L</td><td>8 &micro;L</td><td></td></tr>
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3.5.6 We detected band<br>
−
<tr><td>RNA solution</td><td></td><td></td><td>6 &micro;L</td></tr>
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1.2.4.7.8 We detected no band
−
<tr><td>pure water</td><td></td><td></td><td>2 &micro;L</td></tr>
+
<br>
−
<tr><td>Oligo(dT)<sub>15</sub>primer</td><td>1 &micro;L</td><td>1 &micro;L</td><td>1 &micro;L</td></tr>
+
<br>
−
<tr><td>Random primer</td><td>1 &micro;L</td><td>1 &micro;L</td><td>1 &micro;L</td></tr>
+
there is a band in the circular mRNA fraction which used exo-RNase. This meanes that circular mRNA exists.  
−
<tr><td>total</td><td>10 &micro;L</td><td>10 &micro;L</td><td>10 &micro;L</td></tr>
+
−
</table>
+
−
<br><br>
+
−
Incubate them at 70 &deg;C for 5 minutes.<br>
+
−
Incubate them at 4 &deg;C for 5 minutes.<br>
+
−
Incubate them on ice.<br>
+
−
Mix the following reagents.
+
−
<table class="table" border="2">
+
−
<tr><td>Nuclease Free Water</td><td>4.58 &micro;L</td></tr>
+
−
<tr><td>GoScript&trade; 5&times;reaction buffer</td><td>12.2 &micro;L</td></tr>
+
−
<tr><td>25mM MgCl<sub>2</sub></td><td>6.1 &micro;L</td></tr>
+
−
<tr><td>10mM PCR Nucleotide Mix</td><td>3.05 &micro;L</td></tr>
+
−
<tr><td>Recombinant RNasin Ribo nuclease Inhibitor</td><td>1.52 &micro;L</td></tr>
+
−
<tr><td>GoScript&trade; Reverse</td><td>3.05 &micro;L</td></tr>
+
−
</table>
+
 
<br>
 
<br>
−
Add 10 &micro;L of it each to group1,2,3.<br>
 
−
Synthesize cDNA.<br>
 
−
<table class="table" border="2">
 
−
<tr><td>Annealing</td><td>At 25 &deg;C for 5 minutes</td></tr>
 
−
<tr><td>Elongation</td><td>At 42 &deg;C for 60 minutes</td></tr>
 
−
<tr><td>Inactivation</td><td>At 70 &deg;C for 15 minutes</td></tr>
 
−
<tr><td></td><td>At 4 &deg;C for &infin;</td></tr>
 
−
</table>
 
−
Mix the following reagents.
 
−
<table class="table" border="2">
 
−
<tr><td></td><td colspan="2">Group1</td><td colspan="2">Group2</td><td colspan="2">Group3</td>
 
−
<td colspan="2">Group4 (total RNA without reverse transcription)</td></tr>
 
−
<tr><td></td><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td></tr>
 
−
<tr><td>Nuclease Free Water</td><td>10 &micro;L</td><td>10 &micro;L</td><td>10 &micro;L</td>
 
−
<td>10 &micro;L</td><td>10 &micro;L</td><td>10 &micro;L</td><td>10 &micro;L</td><td>10 &micro;L</td></tr>
 
−
<tr><td>GoTaq 2&times;mix</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td>
 
−
<td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td></tr>
 
−
<tr><td>primer Fw (to detect circular mRNA)</td><td></td><td>2.5 &micro;L</td><td></td><td>2.5 &micro;L</td><td></td><td>2.5 &micro;L</td><td></td>
 
−
<td>2.5 &micro;L</td></tr>
 
−
<tr><td>primer Rv (to detect circular mRNA)</td><td></td><td>2.5 &micro;L</td><td></td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td></td><td>2.5 &micro;L</td>
 
−
<td>2.5 &micro;L</td></tr>
 
−
<tr><td>primer Fw (to detect liner(endogenous) mRNA)</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td><td>2.5 &micro;L</td>
 
−
<td>2.5 &micro;L</td></tr>
 
−
cDNA solution  10ul
 
  
−
</table>
+
<h3>the sequence of Circular mRNA</h3>
 +
<h4>summary of the experiment</h4>
 +
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.<br>
 +
[[File:gifupartreg3.png|500px|]]
 +
<br>
 +
<b>Figure 4. Joint sequence = the evidence of circularization</b>
 +
<br>
 +
<br>
  
 +
<h4>Result</h4>
 +
[[File:gifupartreg4.png|600px|]]
 +
<br>
 +
<b>Figure 5. The evidence of circularization</b>
 +
<br>
 +
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.
 +
 +
<br>
 +
 +
<h2>Synthesis of long-chain proteins</h2>
 +
<h4>Summary of the experiment</h4>
 +
Confirm repeating translation by SDS-PAGE([http://2014.igem.org/Team:Gifu/Protocol#SDS protocol]).<br>
 +
 +
 +
<h4>Result</h4>
 +
[[File:gifupartreg5.png|500px|]]<br>
 +
[[File:gifupartreg6.png|500px|]]<br>
 +
<b>Figure 6. The result of SDS-PAGE</b>
 +
 +
 +
<br>
 +
 +
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.
 +
 +
<br>
 +
<br>
 +
 +
 +
<h2>Derived from RFP</h2>
 +
<h4>Summary of the experiment</h4>
 +
Perform the Western blotting using anti RFP antibody conjugated with peroxidase.<br>
 +
<h4>Result</h4>
 +
[[File:Gifupartreg7.png|200px|]]<br>
 +
<b>Figure 7. The result of the Western blotting </b>
 +
<br>
 +
The proteins over 250 kDa were bound with the antibody. It means that the long-chain protein derives from the RFP.
 +
<br>
 +
 +
<h2>Circularize efficiency</h2>
 +
<h4>Summary of the experiment</h4>
 +
Reverse-transcribe the specific four fragments of DNA(A-D) and calculate the efficiency of　mRNA circularization by the MPN-PCR.
 +
<h4>Result</h4>
 +
The efficiency of circularization was 2.5%.<br>
 +
[http://2014.igem.org/Team:Gifu/Modeling Read more on the modeling.]
 +
 +
 +
<h2>Quantitative determination of proteins</h2>
 +
<h4>Summary of the experiment</h4>
 +
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.
 +
<h4>Result</h4>
 +
[[File:quantitative determination of protein.png|700px|]]<br>
 +
<b>Figure 8. The strength of bands of monomer RFP</b>
 +
<br>
 +
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.
 +
<br>
 +
 +
[[File:concentration of polymer and monomer.png|500px|]]<br>
 +
<b>Table 1. The result that concentration of polymer and monomer </b>
 +
<br>
 +
Concentration of the monomer RFP was 0.57(mg/mL), and the polymer RFP was 0.41(mg/mL).
 +
<br>
 +
<br>
 +
<h3>discussion</h3>
 +
ratio of existence about Circular mRNA and Linear mRNA and concentration of proteins in the same <i>E. coli</i> show as follow.
 +
[[File:efficiency and concentration.png|500px|]]
 +
<br>
 +
<b>Figure 9. Ratio of existence about Circular mRNA and Linear mRNA and concentration of proteins</b>
 +
<br>
 +
When the amount of Circular mRNA is about the same as Linear mRNA,
 +
<br>
 +
[[File:efficiency and concentration2.png|600px|]]<br>
 +
<b>Figure 10. The difference of efficiency between Circular mRNA and Linear mRNA</b>
 +
<br>
 +
Polymer RFP is 27 times as much weight as Monomer RFP.<br>
 +
Circular mRNA synthesized more proteins than Linear mRNA did.
 +
 +
 +
<h2>The ability of coloration</h2>
 +
[[File:RFPGIFU.png|500px|]]<br>
 +
<b>Figure 11. The difference of coloration </b>
 +
<br>
 +
<div id="result2">
 +
1.RFP from linear RNA (with stop codon)<br>
 +
2.RFP from circular RNA (with stop codon)<br>
 +
3.RFP from circular RNA (without stop codon)：using this device<br>
 +
4.RFP from circular RNA (with the stop codon of mRNA circular device)
 +
</div>
 +
The RFP (+histidine tag) polymer didn’t show the fluorescence.<br>
 +
Possible factor <br>
 +
1.The RFP polymer is too huge, so it becomes an inclusion body.<br>
 +
2.The repetitive amino acid sequences are too close, so the conformation of the RFP polymer is in disorder.<br>
 +
 +
<br><br>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 20:27, 2 November 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 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.


Gifu RFP generator.png
Figure 1.In the case that RFP is inserted as protein coding.
Link:

  • An RFP which we use is BBa_K1332002. That's an RFP which combines with a Histidine tag.
  • BBa_K1332011:Histidine tag (8 AA) and RFP semi-permanent generator

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.
Gifupartreg1.png
Figure 2.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

Gifupartreg2.png
Figure 3. 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.
Gifupartreg3.png
Figure 4. Joint sequence = the evidence of circularization

Result

Gifupartreg4.png
Figure 5. The evidence of circularization
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

Gifupartreg5.png
Gifupartreg6.png
Figure 6. The result of SDS-PAGE



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

Gifupartreg7.png
Figure 7. The result of the Western blotting
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

Quantitative determination of protein.png
Figure 8. The strength of bands of monomer RFP
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 polymer and monomer.png
Table 1. The result that concentration of polymer and monomer
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. Efficiency and concentration.png
Figure 9. Ratio of existence about Circular mRNA and Linear mRNA and concentration of proteins
When the amount of Circular mRNA is about the same as Linear mRNA,
Efficiency and concentration2.png
Figure 10. The difference of efficiency between Circular mRNA and 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

RFPGIFU.png
Figure 11. The difference 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


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]