Difference between revisions of "Part:BBa K914008"

(→‎Characterization)
(→‎Characterization)
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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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We performed a set of experiments to show that I-SceI meganuclease is expressed and active in eliminating restriction-site harbouring plasmid.
  
 
====Experimental setup====
 
====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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Firstly, we transformed two plasmids with different antibiotic resistances into NEB Turbo <i>E.Coli</i> strain:
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#<b>First plasmid:</b> Version of the low copy plasmid with encoded generator to express I-SceI meganucllease regulated by pRha.
 
#* Backbone: pSB3C5
 
#* Backbone: pSB3C5
 
#* Resistance: Chloramphenicol
 
#* Resistance: Chloramphenicol
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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.
 
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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{|align="center"
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|-valign="top"
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|[[Image:Paris_Bettencourt_2012_RG_Cm_112TUD_2.jpg|thumb|250px|center|Selection: Cm]]
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|[[Image:Paris_Bettencourt_2012_RG_Amp_112TUD_2.jpg|thumb|250px|center|Selection: Amp]]
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|[[Image:Paris_Bettencourt_2012_RG_AmpCm_112TUD_2.jpg|thumb|250px|center|Selection: Cm & Amp]]
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|}
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 16:57, 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 that I-SceI meganuclease is expressed and active in eliminating restriction-site harbouring plasmid.

Experimental setup

Firstly, we transformed two plasmids with different antibiotic resistances into NEB Turbo E.Coli strain:

  1. First plasmid: Version of the low copy plasmid with encoded generator to express I-SceI meganucllease regulated by pRha.
    • 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.

Selection: Cm
Selection: Amp
Selection: Cm & Amp

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]