Difference between revisions of "Part:BBa K2308016"

 
 
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<partinfo>BBa_K2308016 short</partinfo>
 
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This part contains LacIq repressor, LacIq spacer, LacIq promotor and LacIq coding sequence and is used for Rhodobacter sphaeroides, cells with the device can carry out inducible expression of the target proteins.
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This part is an improvement of [[Part:BBa_V1003]]
  
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===Usage and Biology===
 
  
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This part contains LacIq repressor, LacIq spacer, LacIq promotor and LacIq coding sequence and is used for <i>Rhodobacter sphaeroides 2.4.1</i>, cells with the device can carry out inducible expression of the target proteins.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2308016 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2308016 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2308016 parameters</partinfo>
  
  
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===Functional Parameters===
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<partinfo>BBa_K2308016 parameters</partinfo>
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<p>sYFP2(BBa_K2308003) is the key part in our experiment. It is used both for fusion expression (see BBa_K2308002) and inducible cytoplasm expression (combined with BBa_K2308016), and the host cell is <i>Rhodobacter sphaeroides 2.4.1</i>. </p><br>
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<img src="https://static.igem.org/mediawiki/2017/4/47/F1-D.png" style="height: 400px;">
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<img src="https://static.igem.org/mediawiki/2017/f/fa/F1-E.png" style="height: 400px;">
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<center><p>Figure 1：PCR test of BBa_K2308003(optimized sYFP2) and original sYFP2</p></center><br><br>
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<p> In inducible cytoplasm expression experiment, we constructed the inducible plasmid pIND4 using part BBa_K2308016 and succeeded in turning the <i>Rhodobacter sphaedoides 2.4.1</i>  into inducible strains.IPTG was added （800 μM in final volume）when the OD<sub>700</sub> of the strain was about 0.4（grown for about 24h）.</p>
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                <img src="https://static.igem.org/mediawiki/parts/8/89/LacIq.png" style="width: 600px;">
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<center><p>Figure 2：Confirmed map of plasmid pIND4</p></center>
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<img src="https://static.igem.org/mediawiki/2017/b/be/Cwj_yingguang0002.jpg" style="width: 400px;">
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<img src="https://static.igem.org/mediawiki/2017/5/57/Cwj_yingguang0004.jpg" style="width: 400px;">
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<center><p>Figure 3：fluorescent image of sYFP2 (original) and sYFP2(optimized)</p></center>
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<div class="row" align="left" style="margin-left:10%;">
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<img src="https://static.igem.org/mediawiki/2017/9/99/F5-A%281%29.png" style="width: 400px;">
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<img src="https://static.igem.org/mediawiki/2017/9/99/F5-A%281%29.png" style="width: 400px;">
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<center><p>Figure 4：Fluorescence intensity of WT, sYFP2(optimized), sYFP2(unoptimized) strains.</p></center>
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<br><br>
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<p> It is obvious that after the codon optimization, sYFP2 can be better expressed in <i>Rhodobacter sphaeroides 2.4.1</i>, and the LacIq promotor also worked very well.</p>
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</html>

Latest revision as of 11:51, 1 November 2017


LacIq

This part is an improvement of Part:BBa_V1003


This part contains LacIq repressor, LacIq spacer, LacIq promotor and LacIq coding sequence and is used for Rhodobacter sphaeroides 2.4.1, cells with the device can carry out inducible expression of the target proteins.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 84
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


sYFP2(BBa_K2308003) is the key part in our experiment. It is used both for fusion expression (see BBa_K2308002) and inducible cytoplasm expression (combined with BBa_K2308016), and the host cell is Rhodobacter sphaeroides 2.4.1.


Figure 1：PCR test of BBa_K2308003(optimized sYFP2) and original sYFP2



In inducible cytoplasm expression experiment, we constructed the inducible plasmid pIND4 using part BBa_K2308016 and succeeded in turning the Rhodobacter sphaedoides 2.4.1 into inducible strains.IPTG was added （800 μM in final volume）when the OD700 of the strain was about 0.4（grown for about 24h）.

Figure 2：Confirmed map of plasmid pIND4

Figure 3：fluorescent image of sYFP2 (original) and sYFP2(optimized)



Figure 4：Fluorescence intensity of WT, sYFP2(optimized), sYFP2(unoptimized) strains.



It is obvious that after the codon optimization, sYFP2 can be better expressed in Rhodobacter sphaeroides 2.4.1, and the LacIq promotor also worked very well.