Part:BBa_K3634013:Design
ccdAB promoter + operator
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
As aforementioned, the 113bp ccdAB promoter/operator region merges with the ccdA gene 87bp through the 5' end of the regulatory region. If included in full, all 8 ccdA binding sites would be part of the sequence allowing for maximum control efficiency of gene output (Vandervelde et al., 2017). However, as a RBS was predicted to be present before the ccdA gene, a truncated malfunctioning ccdA protein may well have been expressed. This would have resulted in an unwanted addition to cell metabolic burden and the truncated ccdA may have disrupted binding of functional ccdA to the operator sites.
As 5/8 ccdA binding sites remained unidentified in the 113bp region, it was predicted that removing the ccdA gene sequence would remove a maximum of 3 additional ccdA binding sites based on the observed length of the operator sequences and the spacer regions that existed between the three already identified regions. By the assumption that 5 binding sites would be present in the remaining 86bp promoter/operator region and comparison with Figure 4c of Vandervelde et al. (2017), it was concluded that the efficiency of repression by ccdA would remain high enough to warrant the use of the truncated promoter/operator region in our design.
With regard to the unknown RBS region, adding a weak RBS from the Anderson RBS catalogue on top of the pre-existing sequence would ensure sufficient expression of the output gene.
Source
The ccdAB operon is native to the F plasmid of E.coli. The specific sequence detailed for this part was obtained from Vandervelde et al. (2017) in 'Molecular Mechanisms Governing Ratio-Dependent Transcription Regulation in the CcdAB Operon'.
References
Tam J.E., Kline B.C. 1989. Control of the ccd operon in plasmid F. J. Bacteriol. 171: p2353–2360.
Vandervelde A., Drobnak I., Hadži S., Sterckx Y.GJ., Welte T., Greve. H.D., Charlier D., Efremov R., Loris R., Lah J. 2017. Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon. NAR. 45(6): p2937-2950.