Part:BBa_K879917

LacO ori

This is a plasmid backbone that was designed to have its copy number (number of copies of the plasmid in one cell) controlled by the presence or absence of the inducer molecule Isopropyl β-D-1-thiogalactopyranoside (IPTG). This copy number in turn corresponds to the expression level of the genes on the plasmid.

Usage and Biology

This biobrick has been tested in a number of experiments, outlined below.



Top Right: The results of a plasmid loss experiment. Bacterial cultures were grown at 37^oC in LB broth overnight under selective conditions that favoured plasmid maintenance [chloramphenicol (0.3 uL/mL) and either IPTG (0.05 mM) or ATC (1.07 nM)]. As a starting point, the number of viable cells in each culture was determined by spotting 5 uL of 10-fold serial dilutions ranging from undiluted to 10^-6 (7 spots/culture) onto LB-agar plates containing chloramphenicol and either IPTG or ATC at the concentrations cited above. Cell count/mL of culture was determined by counting (or estimating) the number of colonies at the highest resolvable dilution and multiplying by the dilution factor. Fresh cultures were then made by inoculating one uL of the original cultures into 5 mL of LB broth under conditions that favoured plasmid loss (no antibiotic or inducer, -/-) or plasmid maintenance (with inducer but no antibiotic, +/-; or with both, +/+) and grown as above. The number of viable cells remaining under non-selective conditions were determined as described above.
Top Left: An experiment done with cells plated with this plasmid on a gradient of IPTG centered on the plate. It shows the inability of the cells to survive antibiotic selection without IPTG present.
Bottom Left: One of the possible usages of this plasmid as a cloning method.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2051
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2057
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2051
  Illegal XhoI site found at 1035
  Illegal XhoI site found at 1927
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2051
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2051
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2066
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.