BioBuilder by Natalie Kuldell Rachel Bernstein Karen Ingram & Kathryn M. Hart

Author:Natalie Kuldell, Rachel Bernstein, Karen Ingram & Kathryn M. Hart
Language: eng
Format: epub
Publisher: O'Reilly Media, Inc.
Published: 2015-07-14T16:00:00+00:00

Inside the Black Box

If you search the Registry of Standard Biological Parts, you’ll find any number of inverter devices. Most of them use transcriptional repression to implement the NOT gate logic. In general terms, these inverter devices are composed of several simple parts beginning with a repressor protein such as a lambda repressor, lac repressor, or tet repressor followed by a repressible promoter that’s recognized by the encoded repressor protein, for example, a lambda phage promoter when a lambda repressor is used. If an inverter device is placed between another promoter and its open reading frame (ORF), activation of the upstream promoter now triggers expression of the repressor protein, which recognizes the downstream promoter directing transcription of the ORF, blocking its expression. As a result, the system’s expression pattern is precisely opposite to its behavior in the absence of the inverter.

In the system we’ve been considering here, an inverter was placed between the osmY promoter and the ORF that encodes the wintergreen smell–generating protein. Thus, when the cell is in stationary phase, the osmY promoter is active and so it expresses the inverter. The repressor protein that’s encoded by the inverter is produced, and that repressor binds to the downstream promoter that’s also included in the inverter. The repressed promoter limits transcription of the ORF that’s downstream of the inverter, which is the wintergreen smell–generating protein in the Eau d’coli system. Consider the reverse scenario: when the cells are in log phase, the osmY promoter is not active, the repressor protein encoded by the inverter is not produced, and the promoter directly upstream of the wintergreen smell–generating ORF is active, so the wintergreen scent is produced.

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