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Characterisation of the activity of the sacB BioBrick:

The control strain which does not contain sacB has grown well on both plates, whereas the sacB-containing strain X2 grows poorly on 10% sucrose and E6 does not grow at all. This experiment confirms that sacB is effective as a negative selection marker within the BRIDGE protocol.

Use of sacB for negative selection in liquid culture

The 2011 Wisconsin-Madison team utilized this part in their double selection cassette. It was used to perform directed evolution upon different liquid phase fluorescent E. coli biosensors. In light of their findings, they have suggested improvements to the protocol for use of this part.

For the 2011 Wisconsin-Madison team's project, they required the selection to be done in liquid culture, as opposed to the solid agar plate use shown in the BRIDGE protocol by the 2010 Edinburgh team above. This allowed for testing of a large mutant library that would have been impractical to screen on plates. Additionally, they were testing E. coli biosensors designed to work with liquid analytes which were impossible to use in a solid agar plate (ethanol and alkanes).

As can be seen above, the expression of sacB in the presence of sucrose prevents most growth on a solid plate, albeit with some survivors. In a liquid culture condition, this occasional sucrose-insensitivity presents a greater problem than sporadic colonies on a plate. When sacB-expressing cells were grown overnight in >1% v/v sucrose-containing media, the culture was always turbid in the morning. This was at first thought to reveal that sacB was not being expressed, but a time course plate reader study revealed that was not the case.

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