Difference between revisions of "Part:BBa K2350020"

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<partinfo>BBa_K2350020 short</partinfo>
 
<partinfo>BBa_K2350020 short</partinfo>
  
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===Endolysin-Holin===
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===Holin-Endolysin===
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　　In this part, we combine endolysin (BBa_K2350018) and holin (BBa_K2350019) for cell lysis. Different from the suicide mechanism used by team PeKing (2014 iGEM Beijing), which belongs to &#955; lysis system, the types we used are from T4 phage. Besides, the endolysin and holin both are from iGEM released parts with quality assurance.<br>
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　　This mechanism which contains holin and endolysin was first proposed by 2014 Peking team. They used parts BBa_K1378031(holin) and BBa_K1378032(endolysin) for the suicide mechanism but we decided to choose other parts of holin and endolysin which labeled as BBa_K112000 and BBa_K112806. Moreover, we have also successfully construct the parts which containing both holin and endolysin. <br>
 
　　In this composite part, holin functions as an important regulation of the whole suicide mechanism. Therefore, we choose a lactose-induced promoter, BBa_R0010. As for endolysin, we choose a constitutive promoter, BBa_J23106. In our design, we expect our suicide mechanism can reach maximize effect with this perfect combination.<br>
 
　　In this composite part, holin functions as an important regulation of the whole suicide mechanism. Therefore, we choose a lactose-induced promoter, BBa_R0010. As for endolysin, we choose a constitutive promoter, BBa_J23106. In our design, we expect our suicide mechanism can reach maximize effect with this perfect combination.<br>
  
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&#12288;&#12288;For more details of the connected promoter, RBS, and terminator, you can see in BBa_K2350018 for endolysin part and BBa_K2350019 for holin part.
 
&#12288;&#12288;For more details of the connected promoter, RBS, and terminator, you can see in BBa_K2350018 for endolysin part and BBa_K2350019 for holin part.
  
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===Result===
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Figure 1 is restriction enzyme check electrophoresis result of holin-endolysin construct.
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The marker is 1kb. We use XmaI to digest the sample. The total length is 3843bp as expected.
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<b>Figure 1 </b>
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　　　　[[File:T--NYMU-Taipei—Holin-Endolysin-REcheck.png|200px]]
  
 
===Functional Test===
 
===Functional Test===
 
　　The line graph below presents the general trend of O.D.600 value per hour after lactose is added.<br>
 
　　The line graph below presents the general trend of O.D.600 value per hour after lactose is added.<br>
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　　As the figure shows, the suicide mechanism is induced immediately when lactose is added into the samples. Besides, we can see that the lactose concentration and the OD value of the bacterium with endolysin-holin construct are positively correlated, which means our suicide mechanism does work.
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　　As the figure shows, the suicide mechanism is induced immediately when lactose is added into the samples. Besides, we can see that the lactose concentration and the OD value of the bacterium with holin-endolysin construct are positively correlated, which means our suicide mechanism does work. <br>
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[[File:BBa K2350020 LineGraph.png|700px]]
  
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[[File:BBa K2350020 LineGraph.png|thumb|left]]
 
  
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<>
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<partinfo>BBa_K2350020 short</partinfo>
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<img src="https://parts.igem.org/File:BBa_K2350020_LineGraph.png#file" width="590" height="447" />
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<partinfo>BBa_K2350020 SequenceAndFeatures</partinfo>

Latest revision as of 02:28, 28 October 2017


R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012

Holin-Endolysin

　　This mechanism which contains holin and endolysin was first proposed by 2014 Peking team. They used parts BBa_K1378031(holin) and BBa_K1378032(endolysin) for the suicide mechanism but we decided to choose other parts of holin and endolysin which labeled as BBa_K112000 and BBa_K112806. Moreover, we have also successfully construct the parts which containing both holin and endolysin.
　　In this composite part, holin functions as an important regulation of the whole suicide mechanism. Therefore, we choose a lactose-induced promoter, BBa_R0010. As for endolysin, we choose a constitutive promoter, BBa_J23106. In our design, we expect our suicide mechanism can reach maximize effect with this perfect combination.


Promoter, RBS, and Terminator

　　For more details of the connected promoter, RBS, and terminator, you can see in BBa_K2350018 for endolysin part and BBa_K2350019 for holin part.


Result

Figure 1 is restriction enzyme check electrophoresis result of holin-endolysin construct. The marker is 1kb. We use XmaI to digest the sample. The total length is 3843bp as expected.


Figure 1

　　　　T--NYMU-Taipei—Holin-Endolysin-REcheck.png

Functional Test

　　The line graph below presents the general trend of O.D.600 value per hour after lactose is added.
　　As the figure shows, the suicide mechanism is induced immediately when lactose is added into the samples. Besides, we can see that the lactose concentration and the OD value of the bacterium with holin-endolysin construct are positively correlated, which means our suicide mechanism does work.

BBa K2350020 LineGraph.png


R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1035
    Illegal NheI site found at 1058
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1362
    Illegal AgeI site found at 1432
  • 1000
    COMPATIBLE WITH RFC[1000]