Part:BBa_K590014

pGA3K3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert

This is a Gibson Cloning friendly 3K3 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 3K3 vector.

Usage and Biology

This is a medium copy plasmid backbone that confers kanamycin 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.

Below is the gel image of the plasmid amplified with universal pGA backbone primers pGAsuffix_fwd and pGAprefix_rev. The band is near the expected length of 2750 bp.

1kb Ladder (left), pGA3k3 (right)

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. 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.

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 medium copy pGA3K3 plasmid to express GFP at levels between the low and high copy plasmids.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal NheI site found at 1384
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal BglII site found at 2744
  Illegal BamHI site found at 1
  Illegal XhoI site found at 16
  Illegal XhoI site found at 177
  Illegal XhoI site found at 1020
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal AgeI site found at 1470
  Illegal AgeI site found at 1793
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 2498