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 PexaA, 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. Repeated cycles of this are expected to yield a mutant ethanol sensor system with the desired phenotype. This system should work for any one- or two-component system which users wish to improve similarly.
Applications of BBa_K634007
When placed 3' of the promoter portion of a 1- or 2-component sensor, BBa_634007 can be used in the following ways:
Reporter
The fluorescent mCherry-derived (tagRFP) fluoresces red upon induction. This can be useful for visually verifying expression of the cassette is proceeding normally, first order approximations of promoter activity, and (with plate reader analysis) as a marker for quantification of expression. Cell cultures have not appeared visibly red in our experience, but are easy to see when pelleted (it is less obvious in the below picture than it really is in person).
Selection
Sensor systems can be selected for desired induced expression through supplementation of the culture with the sensor's analyte and kanamycin. The kanamycin resistance gene used is the same as provides resistance in plasmids such as pSB4K5. In this part, it contains a highly efficient RBS.
Counter selection
Sensors can also be selected for absence of expression when uninduced through supplementation of the culture with sucrose. The Bacillus subtilis gene sacB has the effect of killing gram negative bacteria in the presence of sucrose. The counter selection process works well on agar plates, but requires slight adaptations to be used in liquid cultures; long periods of growth appear to extremely frequently give rise to sucrose-insensitive mutants. However, for shorter growths, sacB appears to be a viable way to select against leaky transcription, illustrated here:
See the sacB entry for more information about how the 2011 Wisconsin-Madison team made sacB counter selections work in liquid cultures.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 648
Illegal SapI.rc site found at 30
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