Difference between revisions of "Part:BBa K914008"
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=== Characterization ===
 
=== Characterization ===
  
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We performed a set of experiments to show I-SceI is expressed and active in eliminating restriction-site harbouring plasmid.
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====Experimental setup====
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We transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:
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#<b>First plasmid:</b> Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:
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#* Backbone: pSB3C5
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#* Resistance: Chloramphenicol
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#* Origin of Replication: p15a
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#<b>Second plasmid:</b> High copy plasmid with encoded I-SceI restriction site, [https://parts.igem.org/Part:BBa_K175027 K175027]. This biobrick was a kind gift of the [http://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.
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#* Backbone: pSB1AK3
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#* Resistance: Ampicillin and Kanamycin
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#* Origin of Replication: modified pMB1 derived from pUC19
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We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure  its efficiency.
  
 
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Revision as of 16:51, 27 September 2012

Meganuclease I-SceI controlled by pRha

I-SceI homing endonuclease expression is controlled by pRha promoter. Expression is induced with L-rhamnose. I-SceI doesn't have an LVA degradation tag.

Characterization

We performed a set of experiments to show I-SceI is expressed and active in eliminating restriction-site harbouring plasmid.

Experimental setup

We transformed two plasmids with different antibiotic resistances into NEB Turbo E.Coli strain:

  1. First plasmid: Low copy plasmid with encoded generator to express I-SceI meganucllease. Three version with different promoters was tested: I-SceI meganuclease controlled by pBad, pLac and pRha. For all version:
    • Backbone: pSB3C5
    • Resistance: Chloramphenicol
    • Origin of Replication: p15a
  2. Second plasmid: High copy plasmid with encoded I-SceI restriction site, K175027. This biobrick was a kind gift of the [http://2012.igem.org/Team:TU-Delft TUDelft iGEM team] which we further characterized.
    • Backbone: pSB1AK3
    • Resistance: Ampicillin and Kanamycin
    • Origin of Replication: modified pMB1 derived from pUC19

We expected to perform transformation with both plasmids, and plate with two antibiotics in order to select for double transformants. We would then induce I-SceI expression in those clones to measure its efficiency.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]