Difference between revisions of "Part:BBa K567014"
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<partinfo>BBa_K567014 short</partinfo>
 
<partinfo>BBa_K567014 short</partinfo>
  
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T7 promoter-''metG''(mutated). This biobrick is constructed by putting the mutated ''metG'' (Met-RS) under the control of T7 promoter and lac operator. We have cloned ''metG'' from E.coli and have used error-prone PCR to amplify the ''metG''. KanaR gene with start codon substituted for CGA is used to testify the function of mutated ''metG''. When this biobrick and ''metY''-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LB Kana plate.
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T7 promoter-''metG''(mutated). This biobrick is constructed by putting the mutated ''metG'' (Met-RS) under the control of T7 promoter and lac operator. We have cloned ''metG'' from E.coli and have used error-prone PCR to amplify the ''metG''. KanR gene with start codon substituted for CGA is used to testify the function of mutated ''metG''. When this biobrick and ''metY''-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LB Kanamycin plate.
  
 
===Construction of BBa_K567014===
 
===Construction of BBa_K567014===
  
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In order to charge Met to tRNA<sup>Met</sup> with mutated anticodon, we need to deprive MetRS of its anticodon specificity. Directed Evolution Strategy is used. Error-prone PCR is used to introduce random mutations into MetRS.When this part, ''metY''-CGA (BBa_K567016) and KanaR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kana. We screened the MetRS obtained through error-prone PCR using Kana and obtained one target mutant.  
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In order to charge Met to tRNA<sup>Met</sup> with mutated anticodon, we need to deprive MetRS of its anticodon specificity. Directed Evolution Strategy is used. Error-prone PCR is used to introduce random mutations into MetRS.When this part, ''metY''-CGA (BBa_K567016) and KanR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kan. We screened the MetRS obtained through error-prone PCR using Kanamycin and obtained one target mutant.
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===Characterization of BBa_K567015===
 
===Characterization of BBa_K567015===
  
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When this part, ''metY''-CGA (BBa_K567016) and KanaR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kana. This enzyme lost specificity for tRNA<sup>Met</sup> anticodon while maintained aminoacylation ability.  
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When this part, ''metY''-CGA (BBa_K567016) and KanR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kanamycin. This enzyme lost specificity for tRNA<sup>Met</sup> anticodon while maintained aminoacylation ability.  
  
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[[image:11SJTU-initial_codon_result.jpg|frame|center|fig. Growth of ER2566 with a. ''metG''N + ''metY''-CGA, b. ''metG''M + ''metY''-CGA, c. + ''metG''N, d. + ''metG''M. Growth medium (left): LB Kana+Tet. Growth medium (right): LB Kana.]]
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[[image:11SJTU-initial_codon_result.jpg|frame|center|Fig. Growth of ER2566 with a. ''metG''N + ''metY''-CGA, b. ''metG''M + ''metY''-CGA, c. + ''metG''N, d. + ''metG''M. Growth medium (left): LB Kana+Tet. Growth medium (right): LB Kana.]]
  
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Cell growth shows that the cells show Kana resistance only when both modified MetRS (''metG''M) and modified tRNA<sup>Met</sup>(''metY''-CGA) are transformed into the cell, proving that ''metG''M works well.
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Cell growth shows that the cells show Kanamycin resistance only when both modified MetRS (''metG''M) and modified tRNA<sup>Met</sup>(''metY''-CGA) are transformed into the cell, proving that ''metG''M works well.
  
 
For more information concerning this part, please see [http://2011.igem.org/Team:SJTU-BioX-Shanghai 2011 SJTU-BioX-iGEM]
 
For more information concerning this part, please see [http://2011.igem.org/Team:SJTU-BioX-Shanghai 2011 SJTU-BioX-iGEM]
  
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Related Biobrick:
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PT7-''metG''N  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567015 BBa_K567015])
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''metY''-CGA  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567016 BBa_K567016])
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:36, 29 October 2011

PT7-metGM

T7 promoter-metG(mutated). This biobrick is constructed by putting the mutated metG (Met-RS) under the control of T7 promoter and lac operator. We have cloned metG from E.coli and have used error-prone PCR to amplify the metG. KanR gene with start codon substituted for CGA is used to testify the function of mutated metG. When this biobrick and metY-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LB Kanamycin plate.

Construction of BBa_K567014

In order to charge Met to tRNAMet with mutated anticodon, we need to deprive MetRS of its anticodon specificity. Directed Evolution Strategy is used. Error-prone PCR is used to introduce random mutations into MetRS.When this part, metY-CGA (BBa_K567016) and KanR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kan. We screened the MetRS obtained through error-prone PCR using Kanamycin and obtained one target mutant.

Characterization of BBa_K567015

When this part, metY-CGA (BBa_K567016) and KanR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kanamycin. This enzyme lost specificity for tRNAMet anticodon while maintained aminoacylation ability.

Fig. Growth of ER2566 with a. metGN + metY-CGA, b. metGM + metY-CGA, c. + metGN, d. + metGM. Growth medium (left): LB Kana+Tet. Growth medium (right): LB Kana.

Cell growth shows that the cells show Kanamycin resistance only when both modified MetRS (metGM) and modified tRNAMet(metY-CGA) are transformed into the cell, proving that metGM works well.

For more information concerning this part, please see [http://2011.igem.org/Team:SJTU-BioX-Shanghai 2011 SJTU-BioX-iGEM]


Related Biobrick:

PT7-metGN (BBa_K567015)

metY-CGA (BBa_K567016)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2257
    Illegal XbaI site found at 48
    Illegal PstI site found at 1508
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2257
    Illegal PstI site found at 1508
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2257
    Illegal BamHI site found at 1951
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2257
    Illegal XbaI site found at 48
    Illegal PstI site found at 1508
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2257
    Illegal XbaI site found at 48
    Illegal PstI site found at 1508
  • 1000
    COMPATIBLE WITH RFC[1000]