Difference between revisions of "Part:BBa E0032"
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Unspecified.
 
Unspecified.
  
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==Contribution ==
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 +
*'''Group:''' [https://2019.igem.org/Team:HBUT-China# iGEM Team HBUT China 2019]
 +
*'''Author:''' Qiushi Li
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*'''Summary:''' A fluorescence study to characterize this EYFP protein and compare it against GFP and E0032.
 +
 +
===Characterization ===
 +
<p style="text-align:left">
 +
    <strong><span style="font-size:20px;font-family:&#39;Times New Roman&#39;,serif">Aim &amp; Hypothesis</span></strong>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">According to the registry, BBa_E0034 should be more stable than BBa_E0032, so our hypothesis is BBa_E0034 is more stable than BBa_E0032. In order to determine the fluorescence stability of the two parts (BBa_E0034 and BBa_E0032) expression products, the fluorescence/OD600 (Fl./OD600) values according time in every groups were measured. BBa_J23107, from a constitutive promoter family, was chosen as the promoter, and BBa_B0062 and BBa_B0011 were chosen as the terminator. Those parts had successfully worked with RFP, and that’s why we are using them. BBa_J23107 (promoter) and BBa_B0062 and BBa_B0011 (terminator) were ligated in to vector</span> <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">pSB1C3.</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">&nbsp;</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <strong><span style="font-size:20px;line-height:150%;font-family: &#39;Times New Roman&#39;,serif">Methods:</span></strong>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">1. BBa_E0034 and BBa_E0032 were amplificated by PCR. </span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">2. Connect the BBa_E0034 and BBa_E0032 into the plasmid</span> <span style="font-size:16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">pSB1C3 respectively, and transform them to <em>E. coli</em>.</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">3. Pre-experiment.</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">A. culture them in 5 mL LB medium at 37 </span><span style="font-size:16px;line-height:150%;font-family:宋体">℃</span><span style="font-size:16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif"> &nbsp;and wrap the culture tube with tin paper to avoid light influence.</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">B. Use fluorescence microscopy to observe fluorescence signal feedback after overnight incubation to determine its expression.</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">4. Use primitive <em>E. coli</em> (DH5</span><span style="font-size:16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">α</span><span style="font-size:16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">) as negative control, and use <em>E. coli </em>with the same promoter and terminator connected GFP. Culture the <em>E. coli </em>in 5 mL LB medium in three tubes at 37 </span><span style="font-size:16px;line-height:150%;font-family:宋体">℃</span><span style="font-size:16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif"> (with tin paper as light barrier). </span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">5. Detect the protein expression of E0032, E0034 and blank control in <em>E.coli</em> by laser scanning confocal microscope analysis.</span>
 +
</p>
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<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">6. Measure the ABS and fluorescence of <span style="color:black">blank group and experimental groups </span>every 1 hour. The first 3 hours were not diluted. Diluted 5 times from the fourth time. Measure 10 times in total.</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">7. Draw the fluorescence-ABS curve and compare the stability of the two fluorescent proteins.</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">&nbsp;</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <strong><span style="font-size:20px;line-height:150%;font-family: &#39;Times New Roman&#39;,serif">Results</span></strong>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">1. We successfully connected the gene fragment between the promoter and terminator in pSB1C3, and send it for sequencing. The result showed that we correctly connected the gene fragment. </span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">&nbsp;</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">2. The protein expression of E0032, E0034 and blank control in <em>E.coli</em> cells were detected by laser scanning confocal microscope analysis. </span>
 +
</p>
 +
[[File:E0032.png|500px|thumb|E0032 under the laser scanning confocal microscope]]
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[[File:E0034.png|500px|thumb|E0034 under the laser scanning confocal microscope]]
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[[File:Blank_control.png|500px|thumb|Blank control under the laser scanning confocal microscope]]
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<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">The results indicated that both E0032, E0034 and blank control had fluorescence, but the fluorescence is very weak.</span>
 +
</p>
 +
<p>
 +
    <span style="font-family:&#39;Times New Roman&#39;,serif">&nbsp;</span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">3. In order to confirm the expression of these two yellow fluorescent proteins. Another fluorescent protein, green fluorescent protein was ligated in the same vector, with the same promoter and terminator. The fluorescent microplate reader was used to measure the fluorescence of the three bacteria. </span>
 +
</p>
 +
<p style="line-height:150%">
 +
    <span style="font-size: 16px;line-height:150%;font-family:&#39;Times New Roman&#39;,serif">From the curve we can see that using these two promoter and terminator, the expression levels of these two yellow fluorescent proteins are very low, lower than the green fluorescent protein, slightly higher than the blank bacteria. We are not sure if it can express higher using other promoter and terminator. </span>
 +
</p>
 +
<p>
 +
    <br/>
 +
</p>
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[[File:Curve_of_flo.png|500px|thumb|E0032 E0034 GFP Blank control fluorescence to ABS 600nm]]
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[[File:Characterization_EEG.png|500px|thumb|E0032 E0034 GFP  fluorescence compare]]
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<p>
 +
    <strong><span style="font-size:24px;font-family: &#39;Times New Roman&#39;,serif">Conclusion</span></strong>
 +
</p>
 +
<p>
 +
    <span style="font-size:16px;font-family:&#39;Times New Roman&#39;,serif">We can see fluorescence signals under the laser scanning confocal microscope, which tells that we have correctly built the plasmid. However, comparing with GFP, using the same promoter and terminator, E0034 and E0032 has a very low level of expression. Other promoters and terminators are not tried, this conclusion is only based on the results of this experiment. </span>
 +
</p>
 +
<p>
 +
    <br/>
 +
</p>
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_E0032 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_E0032 SequenceAndFeatures</partinfo>

Revision as of 09:10, 20 October 2019


enhanced yellow fluorescent protein derived from A. victoria GFP

Yellow fluorescent protein (EYFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. Annotated mutation cause a Q81L mutation that appears to not be near the active site.


Usage and Biology

Unspecified.

Contribution

  • Group: iGEM Team HBUT China 2019
  • Author: Qiushi Li
  • Summary: A fluorescence study to characterize this EYFP protein and compare it against GFP and E0032.

Characterization

Aim & Hypothesis

According to the registry, BBa_E0034 should be more stable than BBa_E0032, so our hypothesis is BBa_E0034 is more stable than BBa_E0032. In order to determine the fluorescence stability of the two parts (BBa_E0034 and BBa_E0032) expression products, the fluorescence/OD600 (Fl./OD600) values according time in every groups were measured. BBa_J23107, from a constitutive promoter family, was chosen as the promoter, and BBa_B0062 and BBa_B0011 were chosen as the terminator. Those parts had successfully worked with RFP, and that’s why we are using them. BBa_J23107 (promoter) and BBa_B0062 and BBa_B0011 (terminator) were ligated in to vector pSB1C3.

 

Methods:

1. BBa_E0034 and BBa_E0032 were amplificated by PCR.

2. Connect the BBa_E0034 and BBa_E0032 into the plasmid pSB1C3 respectively, and transform them to E. coli.

3. Pre-experiment.

A. culture them in 5 mL LB medium at 37 ℃  and wrap the culture tube with tin paper to avoid light influence.

B. Use fluorescence microscopy to observe fluorescence signal feedback after overnight incubation to determine its expression.

4. Use primitive E. coli (DH5α) as negative control, and use E. coli with the same promoter and terminator connected GFP. Culture the E. coli in 5 mL LB medium in three tubes at 37 ℃ (with tin paper as light barrier).

5. Detect the protein expression of E0032, E0034 and blank control in E.coli by laser scanning confocal microscope analysis.

6. Measure the ABS and fluorescence of blank group and experimental groups every 1 hour. The first 3 hours were not diluted. Diluted 5 times from the fourth time. Measure 10 times in total.

7. Draw the fluorescence-ABS curve and compare the stability of the two fluorescent proteins.

 

Results

1. We successfully connected the gene fragment between the promoter and terminator in pSB1C3, and send it for sequencing. The result showed that we correctly connected the gene fragment.

 

2. The protein expression of E0032, E0034 and blank control in E.coli cells were detected by laser scanning confocal microscope analysis.

E0032 under the laser scanning confocal microscope
E0034 under the laser scanning confocal microscope
Blank control under the laser scanning confocal microscope

The results indicated that both E0032, E0034 and blank control had fluorescence, but the fluorescence is very weak.

 

3. In order to confirm the expression of these two yellow fluorescent proteins. Another fluorescent protein, green fluorescent protein was ligated in the same vector, with the same promoter and terminator. The fluorescent microplate reader was used to measure the fluorescence of the three bacteria.

From the curve we can see that using these two promoter and terminator, the expression levels of these two yellow fluorescent proteins are very low, lower than the green fluorescent protein, slightly higher than the blank bacteria. We are not sure if it can express higher using other promoter and terminator.


E0032 E0034 GFP Blank control fluorescence to ABS 600nm
E0032 E0034 GFP fluorescence compare

Conclusion

We can see fluorescence signals under the laser scanning confocal microscope, which tells that we have correctly built the plasmid. However, comparing with GFP, using the same promoter and terminator, E0034 and E0032 has a very low level of expression. Other promoters and terminators are not tried, this conclusion is only based on the results of this experiment.


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]