Difference between revisions of "Part:BBa K1332008"
 
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<partinfo>BBa_K1332008 short</partinfo>
 
<partinfo>BBa_K1332008 short</partinfo>
  
−
This part consists of a promoter (lacI regulated), the 3&acute; side of the intron in td gene of T4 phage and RBS. The protein coding sequence that is inserted between this device and mRNA circularization device (3’ side) can be circularized. If you circularized the protein coding sequence (Its a stop codon have been removed.) with mRNA circularization device (3’ side) for endless translation, you can get a circular mRNA that is translated semi-permanently.
+
This part consists of a promoter (lacI regulated)([https://parts.igem.org/Part:BBa_R0010 BBa_R0010]), The 3' side of the intron(+exon fragment) from td gene of T4 phage([https://parts.igem.org/Part:BBa_K1332005 BBa_K1332005]) and RBS([https://parts.igem.org/Part:BBa_B0034 BBa_B0034]). The protein coding sequence that is inserted between this device and mRNA circularization device (3’ side) can be circularized. If you circularized the protein coding sequence (Its a stop codon have been removed.) with mRNA circularization device (3´ side) (endless translation)([https://parts.igem.org/Part:BBa_K1332009 BBa_K1332009]), you can get a circular mRNA that is translated semi-permanently.
  
 
<h2>Circular Parts</h2>
 
<h2>Circular Parts</h2>
−
[[File:cirpart1.png|500px|]]<br>
+
[[File:gifu circular part 11.png|500px|]]<br>
 
<b>Figure 1. mRNA circularization device (5'side)</b><br><br>
 
<b>Figure 1. mRNA circularization device (5'side)</b><br><br>
−
Part which surrounded by red closing line is <b>mRNA circularization device (5’side)</b>.(figure 1)<br>
+
<b>link:</b><br>
 +
<ul>
 +
<li>[https://parts.igem.org/Part:BBa_K1332003 K1332003]:The 5´ side of the intron(+exon fragment) from td gene of T4 phage without stop codon</li>
 +
<li>[https://parts.igem.org/Part:BBa_K1332004 K1332004]:The 5' side of the intron(+exon fragment) from td gene of T4 phage</li>
 +
<li>[https://parts.igem.org/Part:BBa_K1332005 K1332005]:The 3' side of the intron(+exon fragment) from td gene of T4 phage</li>
 +
<li>[https://parts.igem.org/Part:BBa_K1332008 K1332008]:mRNA circularization device (5´ side)</li>
 +
<li>[https://parts.igem.org/Part:BBa_K1332009 K1332009]:mRNA circularization device (3´ side) (endless translation)</li>
 +
</ul>
 +
 
 +
A Part surrounded by red closing line is <b>mRNA circularization device (5’side)</b>.(figure 1)<br>
  
 
<h2>How to use</h2>
 
<h2>How to use</h2>
Line 14: Line 23:
 
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)<br>
 
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)<br>
 
[[File:prefix.png|600px|]]<br>
 
[[File:prefix.png|600px|]]<br>
−
<b>Figure 2. Modifying the prefix of a protein coding sequence</b><br><br>
+
<b>Figure 2. Modifying the prefix of a protein coding sequence</b><br><br><br>
 +
 
 
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)<br>
 
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)<br>
 
[[File:suffix.png|500px|]]<br>
 
[[File:suffix.png|500px|]]<br>
−
<b>Figure 3. Modifying the suffix of a protein coding sequence</b><br><br>
+
<b>Figure 3. Modifying the suffix of a protein coding sequence</b><br><br><br>
−
And then after that, you insert the protein coding sequence between this device and <b>mRNA cicularization device (3’ side)</b>. At last, you insert the plasmid into <i>E.coli</i>. (figure 4)<br>
+
−
(3' side device is [https://parts.igem.org/Part:BBa_K1332009 K1332009])<br>
+
−
[[File:howtouse2.png|500px|]]<br>
+
−
<b>Figure 4. How to use Ciucular parts</b><br><br>
+
  
−
<h2>Mechanism</h2>
+
And then after that, you insert the protein coding sequence between this device and <b>mRNA cicularization device (3’ side)(endless translation)</b>. At last, you insert the plasmid into <i>E.coli</i>. (figure 4)<br>
 +
3' side device(endless translation) is [https://parts.igem.org/Part:BBa_K1332009 K1332009]<br>
 +
[[File:gifu howtouse2.png|500px|]]<br>
 +
<b>Figure 4. How to use Ciucular parts</b><br>
 +
<b>Link:</b>
 +
<ul>
 +
<li>[https://parts.igem.org/Part:BBa_K1332008 K1332008]:mRNA circularization device (5´ side)</li>
 +
<li>[https://parts.igem.org/Part:BBa_K1332009 K1332009]:mRNA circularization device (3´ side) (endless translation)</li>
 +
</ul>
 +
<br><br>
  
 +
<h2>Mechanism</h2>
 +
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)<br>
 +
[[File:Gifu mechanism2.png|500px|]]<br>
 
<b>Figure 5. Mechanism of mRNA circularization</b><br><br>
 
<b>Figure 5. Mechanism of mRNA circularization</b><br><br>
  
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Adjust the length of a circular mRNA to multiples of 3 to keep a pattern of the reading frame.<br><br>
 
Adjust the length of a circular mRNA to multiples of 3 to keep a pattern of the reading frame.<br><br>
  
 +
 +
<h1><b>When protein coding is RFP coding, its results shows as follow.</b></h1>
 +
[[File:Gifu RFP generator.png|500px|]]<br>
 +
<b>Figure 7.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>
  
  
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<br>
 
<br>
 
[[File:gifupartreg1.png|500px|]]
 
[[File:gifupartreg1.png|500px|]]
 +
<br>
 +
<b>Figure 8.The difference between Linear RNA and circular RNA in two types of RNase(endo or exo) reaction  </b>
 
<br>
 
<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.
 
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.
Line 58: Line 88:
 
<br>
 
<br>
 
<h4>Result</h4>
 
<h4>Result</h4>
−
[[File:gifupartreg2.png|500px|]]
+
[[File:gifupartreg2.png|500px|]]<br>
 +
<b>Figure 9. Electrophoresis results</b>
  
 
3.5.6 We detected band<br>
 
3.5.6 We detected band<br>
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<h3>the sequence of Circular mRNA</h3>
 
<h3>the sequence of Circular mRNA</h3>
 
<h4>summary of the experiment</h4>
 
<h4>summary of the experiment</h4>
−
To get the evidence of circularization, we determined the sequence of circular mRNA that contains joint. The joint is made after the circularization.<br>
+
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|]]
 
[[File:gifupartreg3.png|500px|]]
 +
<br>
 +
<b>Figure 10. Joint sequence = the evidence of circularization</b>
 +
<br>
 +
<br>
  
 +
<h4>Result</h4>
 +
[[File:gifupartreg4.png|600px|]]
 +
<br>
 +
<b>Figure 11. 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 12. 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 13. 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 14. 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 15. 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 16. 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 17. 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>
 
<br><br>

Latest revision as of 20:18, 2 November 2014

mRNA circularization device (5 side)

This part consists of a promoter (lacI regulated)(BBa_R0010), The 3' side of the intron(+exon fragment) from td gene of T4 phage(BBa_K1332005) and RBS(BBa_B0034). The protein coding sequence that is inserted between this device and mRNA circularization device (3’ side) can be circularized. If you circularized the protein coding sequence (Its a stop codon have been removed.) with mRNA circularization device (3´ side) (endless translation)(BBa_K1332009), you can get a circular mRNA that is translated semi-permanently.

Circular Parts

Gifu circular part 11.png
Figure 1. mRNA circularization device (5'side)

link:

  • K1332003:The 5´ side of the intron(+exon fragment) from td gene of T4 phage without stop codon
  • K1332004:The 5' side of the intron(+exon fragment) from td gene of T4 phage
  • K1332005:The 3' side of the intron(+exon fragment) from td gene of T4 phage
  • K1332008:mRNA circularization device (5´ side)
  • K1332009:mRNA circularization device (3´ side) (endless translation)

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)
Prefix.png
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)
Suffix.png
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)(endless translation). At last, you insert the plasmid into E.coli. (figure 4)
3' side device(endless translation) is K1332009
Gifu howtouse2.png
Figure 4. How to use Ciucular parts
Link:

  • K1332008:mRNA circularization device (5´ side)
  • K1332009:mRNA circularization device (3´ side) (endless translation)



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)
Gifu mechanism2.png
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)
Cyc-seq.png
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.

Gifu RFP generator.png
Figure 7.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 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

Gifupartreg2.png
Figure 9. 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 10. Joint sequence = the evidence of circularization

Result

Gifupartreg4.png
Figure 11. 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 12. 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 13. 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 14. 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 15. 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 16. 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 17. 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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]