Plasmid_Backbone

Part:BBa_K590011

Designed by: Michael Brasino, Alicia Wong, Rashmi Ravichandran   Group: iGEM11_Washington   (2011-09-15)
Revision as of 00:15, 3 November 2011 by Robere (Talk | contribs) (Characterization)

pGA1C3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This Gibson Cloning friendly 1C3 plasmid backbone 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].

Usage and Biology

This is a high copy plasmid backbone which has Chloramphenicol resistance, with pLac promoter and GFP. This is a high efficiency Gibson Assembly vector. 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

A sister plasmid (pGA1A3) with the same BglBrick prefix and suffix regions 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. By averaging results from 6 plates, the Gibson efficiency was determined to be 0.991129032, ~99%! 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. As expected, we found the high copy pGA1C3 plasmid to express GFP at levels comparable to the other high copy plasmid pGA1A3.

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 2051
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2051
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2051
    Illegal BglII site found at 2066
    Illegal BamHI site found at 1
    Illegal XhoI site found at 16
    Illegal XhoI site found at 1035
    Illegal XhoI site found at 1927
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2051
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2051
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


[edit]
Categories
Parameters
copies100-300
originpMB1
resistanceChloramphenicol