Difference between revisions of "Part:BBa K567013"
(→‎Characterization of BBa_K567013)
 
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<partinfo>BBa_K567013 short</partinfo>
 
<partinfo>BBa_K567013 short</partinfo>
  
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tRNA(Asp) with its anticodon mutated to CUA (base pairing stop codon UAG) and under the control of lpp promoter. This biobrick is constructed first by cloning the tRNA(Asp) from AspV in E.coli, then the anticodon region is site-directed mutated.
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tRNA(Asp) with its anticodon mutated to CUA (base pairing stop codon UAG) and under the control of ''aspV'' promoter. This biobrick is constructed first by cloning the tRNA(Asp) from ''aspV'' in E.coli, then the anticodon region is site-directed mutated.
  
  
 
===Construction of BBa_K567013===
 
===Construction of BBa_K567013===
  
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This biobrick is constructed first by cloning the tRNA<sup>Asp</sup> from ''AspV'' in ''E.coli'', then the anticodon region is site-directed mutated. This part is constructed on the backbone plasmid pACYC184.
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This biobrick is constructed first by cloning the tRNA<sup>Asp</sup> from ''aspV'' in ''E.coli'', then the anticodon region is site-directed mutated. This part is constructed on the backbone plasmid pACYC184.
  
 
===Characterization of BBa_K567013===
 
===Characterization of BBa_K567013===
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This part acts as a stop codon suppressor tRNA. It can be charged with Asp.  
 
This part acts as a stop codon suppressor tRNA. It can be charged with Asp.  
  
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We have used P''bla''-Luc-TAG as our Reporter. The amount of luciferase produced is reflected using the bioluminescence emitted during the luciferin reaction. Our results demonstrate that this part can suppress TAG insertion into luciferase. In the experimental group, with the help of BBa_K567011 PT7-TDRS (Favorite Part), luciferase-TAG was produced and bioluminescence was emitted during the luciferin reaction. '''These results proved that this tRNA<sup>Asp</sup> can effectively suppress stop codon.  
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We have used P''bla''-Luc-TAG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567003 BBa_K567003]) as our Reporter. The amount of luciferase produced is reflected using the bioluminescence emitted during the luciferin reaction. Our results demonstrate that TAG insertion into luciferase blocks luciferase production, which was shown in the control group. In the experimental group, with the help of PT7-TDRS([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011]) and tRNA<sup>Asp</sup>-TAG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567013 BBa_K567013]),luciferase was produced and bioluminescence was emitted during the luciferin reaction. These results proved that '''Stop-Codon Switch can turn on protein expression.'''
  
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[[image:11SJTU-stop-codon_switch.jpg|frame|center|fig. Functional Analysis of Stop-Codon Switch. ER2566 cannot produce luciferase with BBa_K567003 Pbla-Luc-TAG only. When BBa_K567011 PT7-TDRS (Favorite Part) and BBa_K567013 tRNA<sup>Asp</sup>-TAG (Favorite Part) are also transformed into the cell, luciferase is produced. The results proved Stop-Codon Switch as a strict molecular switch without background noise.This tRNA<sup>Asp</sup> is part of the Stop-Codon Switch]]
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[[image:11SJTU_stop_02.jpg|thumb|600px|center|''Fig.1'' Functional Analysis of Stop-Codon Switch. ER2566 cannot produce luciferase with Pbla-Luc-TAG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567003 BBa_K567003]) only. When PT7-TDRS ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011]) and tRNA<sup>Asp</sup>-TAG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567013 BBa_K567013]) are also transformed into the cell, luciferase is produced. The results proved Stop-Codon Switch as a strict molecular switch without background noise.]]
  
  
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Related Biobrick:
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PT7-TDRS  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011])
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 02:21, 29 October 2011

tRNA(Asp)-TAG

tRNA(Asp) with its anticodon mutated to CUA (base pairing stop codon UAG) and under the control of aspV promoter. This biobrick is constructed first by cloning the tRNA(Asp) from aspV in E.coli, then the anticodon region is site-directed mutated.


Construction of BBa_K567013

This biobrick is constructed first by cloning the tRNAAsp from aspV in E.coli, then the anticodon region is site-directed mutated. This part is constructed on the backbone plasmid pACYC184.

Characterization of BBa_K567013

This part acts as a stop codon suppressor tRNA. It can be charged with Asp.

We have used Pbla-Luc-TAG (BBa_K567003) as our Reporter. The amount of luciferase produced is reflected using the bioluminescence emitted during the luciferin reaction. Our results demonstrate that TAG insertion into luciferase blocks luciferase production, which was shown in the control group. In the experimental group, with the help of PT7-TDRS(BBa_K567011) and tRNAAsp-TAG(BBa_K567013),luciferase was produced and bioluminescence was emitted during the luciferin reaction. These results proved that Stop-Codon Switch can turn on protein expression.

Fig.1 Functional Analysis of Stop-Codon Switch. ER2566 cannot produce luciferase with Pbla-Luc-TAG (BBa_K567003) only. When PT7-TDRS (BBa_K567011) and tRNAAsp-TAG (BBa_K567013) are also transformed into the cell, luciferase is produced. The results proved Stop-Codon Switch as a strict molecular switch without background noise.


Related Biobrick:

PT7-TDRS (BBa_K567011)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 281
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 281
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 281
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 281
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 281
  • 1000
    COMPATIBLE WITH RFC[1000]