Difference between revisions of "Part:BBa K590010"
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===Usage and Biology===
 
===Usage and Biology===
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This is a medium copy plasmid backbone that confers kanamycin resistance. It was deposited in the registry with an insert coding for [https://parts.igem.org/wiki/index.php?title=Part:BBa_I7107 LacI-repressible GFP].
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This is a high copy plasmid backbone that confers ampicillin resistance. It was deposited in the registry with an insert coding for [https://parts.igem.org/wiki/index.php?title=Part:BBa_I7107 LacI-repressible GFP]. This plasmid has been altered to conform to [http://dspace.mit.edu/handle/1721.1/46747 RFC 21] standards, with arbitrary, Gibson-friendly sequences placed in between the restriction enzyme sites EcoRI-BglII, and BamHI-PstI.
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Below is the gel image of the plasmid amplified with universal pGA backbone primers [https://parts.igem.org/Part:BBa_K590086 pGAsuffix_fwd] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590084 pGAprefix_rev]. The band is near the expected length of 2750 bp.
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[[Image:Igem2011 3k3 placGFP gel.png|thumb|center|1kb Ladder (left), pGA3k3 (right)]]
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===Characterization===
 
===Characterization===
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This plasmid has been shown to have much higher efficiency than the equivalent pSB vector. See [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults#Comparison_between_pGA_and_pSB_vectors Gibson Assembly efficiency assay] page for details on the protocol and efficiency measurements.
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This plasmid has been shown to have much higher efficiency than the equivalent pSB vector. We determined the cloning efficiency by dividing the # of bright colonies by the (# of total colonies - # of background colonies). The background colonies were determined by a control sample containing 100 picograms of  just the pGA backbone.  See [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults#Comparison_between_pGA_and_pSB_vectors Gibson Assembly efficiency assay] page for details on the protocol and efficiency measurements.
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<center>https://static.igem.org/mediawiki/2011/d/d1/Washington_pGAefficiency_summary.jpg</center>
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<center>https://static.igem.org/mediawiki/2011/d/d1/Washington_pGAefficiency_summary.jpg</center>  
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To understand how the copy number varies from plasmid to plasmid, the pGA vectors were transformed into a <i>lacI</i> knockout (strain [http://cgsc.biology.yale.edu/Strain.php?ID=16959 2.320]) and the fluorescence levels were measured by flow cytometry. It was found that the fluorescence level is highest in the high copy plasmids:
  
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<center> [[Image:Washington 2011 pGA vector fluorescence means v2.pdf|400px]] </center>
  
 
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Latest revision as of 00:00, 3 November 2011

pGA1A3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This is a Gibson Cloning friendly 1A3 plasmid backbone that was made by [http://2011.igem.org/Team:Washington UW iGEM Team 2011] as part of the [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Assembly toolkit]. Using this plasmid increases the efficiency for anyone doing Gibson cloning into a 1A3 vector.

Usage and Biology

This is a high copy plasmid backbone that confers ampicillin resistance. It was deposited in the registry with an insert coding for LacI-repressible GFP. This plasmid has been altered to conform to [http://dspace.mit.edu/handle/1721.1/46747 RFC 21] standards, with arbitrary, Gibson-friendly sequences placed in between the restriction enzyme sites EcoRI-BglII, and BamHI-PstI.

Characterization

This plasmid has been shown to have much higher efficiency than the equivalent pSB vector. We determined the cloning efficiency by dividing the # of bright colonies by the (# of total colonies - # of background colonies). The background colonies were determined by a control sample containing 100 picograms of just the pGA backbone. See [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults#Comparison_between_pGA_and_pSB_vectors Gibson Assembly efficiency assay] page for details on the protocol and efficiency measurements.

Washington_pGAefficiency_summary.jpg

To understand how the copy number varies from plasmid to plasmid, the pGA vectors were transformed into a lacI knockout (strain [http://cgsc.biology.yale.edu/Strain.php?ID=16959 2.320]) and the fluorescence levels were measured by flow cytometry. It was found that the fluorescence level is highest in the high copy plasmids:

Washington 2011 pGA vector fluorescence means v2.pdf

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2136
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2136
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2136
    Illegal BglII site found at 2151
    Illegal BamHI site found at 1
    Illegal XhoI site found at 16
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2136
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2136
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 1175