Part:BBa_K634007:Experience

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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.

Issues in practice

sacB appears to be something of a lightning rod for loss of function mutations. As can be seen above, sucrose lethality is only maintained in a population for approximately 5 hours. At this point, we presume that spontaneous mutations in sacB produce sucrose-insensitive mutants.

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