Difference between revisions of "Part:BBa K2308002"
 
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<partinfo>BBa_K2308002 short</partinfo>
 
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This part is used to knock in the gene sYFP2 in Rhodobacter Sphaeroides, replacing the end coden of gene puhA, and sYFP2 will be expressed with H-subunit of light reaction center as fusion protein. The part is composed of 404bp of upstream DNA sequence(the end coden of puhA is not included) and 720bp coding sequence of sYFP2 (Part:BBa_K2308003) and 562bp of downstream DNA sequence. After homologous recombination, sYFP2 will be expressed with the H-subunit through fusion expression.
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This part is used to knock in the gene sYFP2 in <i>Rhodobacter sphaeroides 2.4.1</i>, replacing the end coden of gene puhA, and sYFP2 will be expressed with H subunit of light reaction center as fusion protein. The part is composed of 404bp of upstream DNA sequence(the end coden of puhA is not included) and 720bp coding sequence of sYFP2 (Part:BBa_K2308003) and 562bp of downstream DNA sequence. After homologous recombination, sYFP2 will be expressed with the H subunit through fusion expression.
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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2308002 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K2308002 parameters</partinfo>
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<p>BBa_K2308002 is an important part in our experiment. It is composed of BBa_K2308006，BBa_K2308003 and BBa_K2308007. The function of this part is to carry out the homologous recombination on the genome of <i>Rhodobacter sphaeroides 2.4.1.</i></p><br><br>
 
<p>BBa_K2308002 is an important part in our experiment. It is composed of BBa_K2308006，BBa_K2308003 and BBa_K2308007. The function of this part is to carry out the homologous recombination on the genome of <i>Rhodobacter sphaeroides 2.4.1.</i></p><br><br>
  
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<p>In the experiment, sYFP2 was designed to be fused to the C-terminal of subunit H in the reaction center(RC) of <i>Rhodobacter sphaeroides 2.4.1.</i> </p>
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<p>In fusion experiment, sYFP2 was designed to be fused to the C-terminal of H subunit in the reaction center(RC) of <i>Rhodobacter sphaeroides 2.4.1.</i> </p>
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<img src="https://static.igem.org/mediawiki/2017/5/59/Fusion_protein.gif" alt="">
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<center><img class="featuredparts-img" src="https://static.igem.org/mediawiki/2017/5/59/Fusion_protein.gif" style="width:300px;">
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<center><p style="font-size: 10px;">Figure 1：Modelling of RC-sYFP2 (GIF)</p></center>
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<p style="font-size: 10px;">Figure 1：Modelling of RC-sYFP2 (GIF)</p></center>
 
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<p>In fusion expression experiment: we constructed the plasmid using part BBa_K2308002, and after transformation and gene recombination, single clones are selected by antibiotics and PCR tests. </p>
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<p>We constructed the plasmid using part BBa_K2308002, and after transformation and gene recombination, single clones are selected by antibiotics and PCR tests. In order to achieve gene knock out / knock in, we used a special  plasmid called pDM4. The plasmid pDM4 contains gene <i>Mob</i> replicator <I>oriV</i> and gene <i>SacB</i> .Gene <i>mob</i> can be used to facilitate the use of bonding method of transformation. Replicator oriV couldn't replicate in the globular bacteria (Erythrocytes abscess λπ factor ) , thus single clones could be selected by chl resistance of the plasmid . Finally , through the gene <I>SacB</i> on pDM4 , sucrose was screened for secondary recombination .</p>
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<img src="https://static.igem.org/mediawiki/2017/0/04/F1-B.jpg" alt="">
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<div class="row" align="left" style="margin-left:10%;">
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<center><p style="font-size: 10px;">Figure 2：PCR test of BBa_K2308002</p></center>
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<center><img src="https://static.igem.org/mediawiki/2017/5/52/Ki1.png" style="width: 600px;">
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<center><p style="font-size: 10px;">Figure 2：Confirmed map of part BBa_K2308002</p></center>
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<center><img class="featuredparts-img" src="https://static.igem.org/mediawiki/2017/0/04/F1-B.jpg" style="width:300px;">
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<p style="font-size: 10px;">Figure 3：PCR test of BBa_K2308002</p></center>
 
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<p>Fluorescence image shows sYFP2 which is fused with H submit of <i>Rhodobacter sphaeroides 2.4.1</i> ’s reaction center folded correctly and was able to emit fluorescence. </p>
 
<p>Fluorescence image shows sYFP2 which is fused with H submit of <i>Rhodobacter sphaeroides 2.4.1</i> ’s reaction center folded correctly and was able to emit fluorescence. </p>
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<img src="https://static.igem.org/mediawiki/2017/b/b9/F3.jpg" alt="">
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<center><img class="featuredparts-img" src="https://static.igem.org/mediawiki/2017/b/b9/F3.jpg" style="width:300px;">
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<center><p style="font-size: 10px;">Figure 3：Fluorescence image of whole cells of RC-SYFP2 cells when excited at 495nm</p></center>
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<p style="font-size: 10px;">Figure 4：Fluorescence image of whole cells of RC-SYFP2 cells when excited at 495nm</p></center>
 
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<img src="https://static.igem.org/mediawiki/2017/3/34/F2.png" alt="">
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<center><img class="featuredparts-img" src="https://static.igem.org/mediawiki/2017/e/e7/Absorption_spectrum2.png" style="width:300px;">
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<center><p style="font-size: 10px;">Figure 4：Absorption spectra of RC-sYFP2 strains compared with wild type.</p></center>
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<p style="font-size: 10px;">Figure 5：Absorption spectra of RC-sYFP2 strains compared with wild type.</p></center>
 
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<p> The RC-sYFP2 fusion strain was created in a wild type background. Absorption spectrum of membranes from this strain recorded at room temperature show no isolated sYFP2 peak due to overlap with absorption of carotenoid B at 514 nm.</p>
 
<p> The RC-sYFP2 fusion strain was created in a wild type background. Absorption spectrum of membranes from this strain recorded at room temperature show no isolated sYFP2 peak due to overlap with absorption of carotenoid B at 514 nm.</p>
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<img src="https://static.igem.org/mediawiki/2017/4/42/F4.png" alt="">
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<center><img class="featuredparts-img" src="https://static.igem.org/mediawiki/2017/4/42/F4.png" style="width:300px;">
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<center><p style="font-size: 10px;">Figure 5：Growth rate of WT and WT RC-sYFP2.</p></center>
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<p style="font-size: 10px;">Figure 5：Growth rate of WT and WT RC-sYFP2.</p></center>
 
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<p>The biomass of Rhodobacter sphaeroides 2.4.1 didn’t arise very much since the energy from sYFP2 is too little.</p>
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<p>The biomass of <i>Rhodobacter sphaeroides 2.4.1</i> didn’t arise very much since the energy from sYFP2 is too little.</p>
  
  
  
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</body>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2308002 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K2308002 parameters</partinfo>
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Latest revision as of 21:59, 1 November 2017


sYFP2 knock In for fusion expression

This part is used to knock in the gene sYFP2 in Rhodobacter sphaeroides 2.4.1, replacing the end coden of gene puhA, and sYFP2 will be expressed with H subunit of light reaction center as fusion protein. The part is composed of 404bp of upstream DNA sequence(the end coden of puhA is not included) and 720bp coding sequence of sYFP2 (Part:BBa_K2308003) and 562bp of downstream DNA sequence. After homologous recombination, sYFP2 will be expressed with the H subunit through fusion expression.





Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 319
    Illegal BamHI site found at 1217
    Illegal XhoI site found at 96
    Illegal XhoI site found at 159
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 36
    Illegal NgoMIV site found at 54
    Illegal NgoMIV site found at 336
  • 1000
    COMPATIBLE WITH RFC[1000]




BBa_K2308002 is an important part in our experiment. It is composed of BBa_K2308006，BBa_K2308003 and BBa_K2308007. The function of this part is to carry out the homologous recombination on the genome of Rhodobacter sphaeroides 2.4.1.



In fusion experiment, sYFP2 was designed to be fused to the C-terminal of H subunit in the reaction center(RC) of Rhodobacter sphaeroides 2.4.1.

Figure 1：Modelling of RC-sYFP2 (GIF)



We constructed the plasmid using part BBa_K2308002, and after transformation and gene recombination, single clones are selected by antibiotics and PCR tests. In order to achieve gene knock out / knock in, we used a special plasmid called pDM4. The plasmid pDM4 contains gene Mob replicator oriV and gene SacB .Gene mob can be used to facilitate the use of bonding method of transformation. Replicator oriV couldn't replicate in the globular bacteria (Erythrocytes abscess λπ factor ) , thus single clones could be selected by chl resistance of the plasmid . Finally , through the gene SacB on pDM4 , sucrose was screened for secondary recombination .

Figure 2：Confirmed map of part BBa_K2308002



Figure 3：PCR test of BBa_K2308002



Fluorescence image shows sYFP2 which is fused with H submit of Rhodobacter sphaeroides 2.4.1 ’s reaction center folded correctly and was able to emit fluorescence.

Figure 4：Fluorescence image of whole cells of RC-SYFP2 cells when excited at 495nm



Figure 5：Absorption spectra of RC-sYFP2 strains compared with wild type.



The RC-sYFP2 fusion strain was created in a wild type background. Absorption spectrum of membranes from this strain recorded at room temperature show no isolated sYFP2 peak due to overlap with absorption of carotenoid B at 514 nm.

Figure 5：Growth rate of WT and WT RC-sYFP2.



The biomass of Rhodobacter sphaeroides 2.4.1 didn’t arise very much since the energy from sYFP2 is too little.