Difference between revisions of "Part:BBa K1906005"
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<partinfo>BBa_K1906005 short</partinfo>
 
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Multifunctional protein that promotes group II intron splicing and mobility by acting both on RNA and DNA. It has three activities: reverse transcriptase (RT) for intron duplication, maturase to promote splicing, and DNA endonuclease for site-specific cleavage of recipient alleles. The intron-encoded protein promotes splicing by facilitating the formation of the catalytically active structure of the intron RNA. After splicing, the protein remains bound to the excised intron lariat RNA, forming ribonucleoprotein particles, and cleaving the antisense strand of the recipient DNA in the 3' exon. After DNA cleavage, retrohoming occurs by a target DNA-primed reverse transcription of the intron RNA that had reverse spliced into the sense strand of the recipient DNA. It also contributes to the recognition of the DNA target site and acts as a repressor of its own translation.
 
  
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===Characterlization===
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Whole cell lysate, supernatant and precipitations of cells with different constructs are loaded into SDS-PAGE gels. Figure 3 shows the electrophoresis results of samples that transformed with three different constructs: 1. pETDuet-1-Rep (Expressing β subunit alone) 2. pACYC-TS-TU (co-expressing EF-Ts and EF-Tu) 3. pACYC-TU (Expressing EF-Tu alone) It can be clearly observed that, unpon inducement of IPTG, pETDuet-1-Rep doesn't lead to the abundant production of β subunit. None of the three lanes of cells that transformed with pETDuet-1-Rep display a detectable band at 65.6 kDa, the size of β subunit. The cells that transformed with pACYC-TS-TU and pACYC-TU, however, shows unambiguous production of EF-Ts (position indicated by red arrow) and EF-Tu (position indicated by orange arrow).
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https://static.igem.org/mediawiki/2016/f/f7/11.jpeg
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https://static.igem.org/mediawiki/2016/d/d0/12.jpeg
 
https://static.igem.org/mediawiki/2016/d/d0/12.jpeg

Revision as of 17:00, 20 October 2016


Beta-subunit of Qbeta replicase holoenzyme


Characterlization

Whole cell lysate, supernatant and precipitations of cells with different constructs are loaded into SDS-PAGE gels. Figure 3 shows the electrophoresis results of samples that transformed with three different constructs: 1. pETDuet-1-Rep (Expressing β subunit alone) 2. pACYC-TS-TU (co-expressing EF-Ts and EF-Tu) 3. pACYC-TU (Expressing EF-Tu alone) It can be clearly observed that, unpon inducement of IPTG, pETDuet-1-Rep doesn't lead to the abundant production of β subunit. None of the three lanes of cells that transformed with pETDuet-1-Rep display a detectable band at 65.6 kDa, the size of β subunit. The cells that transformed with pACYC-TS-TU and pACYC-TU, however, shows unambiguous production of EF-Ts (position indicated by red arrow) and EF-Tu (position indicated by orange arrow).

12.jpeg

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1317
    Illegal BamHI site found at 1460
    Illegal XhoI site found at 991
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1473
  • 1000
    COMPATIBLE WITH RFC[1000]