Part:BBa_K634007

Double selection cassette (tagRFP-sacB-kanR)

For additional detailed information, characterization, and usage, see the experience page.

Purpose This device can serve three roles (fluorescent reporter, selectable marker, counter-selectable marker) in an E. coli-based system. The consolidation of these functions to a single device should facilitate directed evolution studies. A sample usage of K634007 is presented below to clarify. This part is intended to be cloned downstream of one- or two-component sensor systems, to be expressed as (or in tandem with) the reporter. The most benefit can be derived from this part in studies involving large mutant libraries of sensor components, such as the product of an error-prone PCR amplification of a sensor kinase.

Use case: 2011 Wisconsin-Madison For the ethanol sensor system composed of the sensor kinase exaD, transcription factor exaE, and the targeted promoter, the UW-Madison 2011 team sought to modify the above parts in order to increase induced expression and decrease leaky expression. Strains were produced with this part 3' of the promoter, and one of the above sensor components knocked out. Mutant libraries were generated using Agilent's GeneMorph II mutagenesis kit. Once cloned, mutants could readily be screened for a first-order approximation of system activity in liquid culture containing ethanol. To eliminate mutants which had a mutation preventing system expression, a selection was performed through growth in the presence of ethanol and kanamycin. Mutants surviving this were then washed and plated to remove residual ethanol, then introduced to a counter-selective media containing sucrose and no ethanol. In this condition, we eliminated mutants which had a mutation causing significant levels of constitutive expression.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 648
  Illegal SapI.rc site found at 30