Difference between revisions of "Part:BBa K3634014:Design"

Revision as of 13:11, 7 August 2020

CcdAB-Controlled mf-Lon Protease

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 the weak BBa_B0031 RBS from the Anderson RBS catalogue on top of the pre-existing sequence would ensure sufficient expression of mf-Lon protease. The mf-Lon protease sequence itself was codon optimised for E. coli using the website www.jcat.de to improve expression efficiency in the chassis organism.

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'. The mf-Lon protease was obtained from BBa_K2333011 which was adapted from Gur & Sauer's 'Evolution of the ssrA degradation tag in Mycoplasma: specificity switch to a different protease' (2008). The part was also codon optimised for E.coli by William & Mary iGEM 2017 using the website www.jcat.de.

References

Tam J.E., Kline B.C. 1989. Control of the ccd operon in plasmid F. J. Bacteriol. 171: p2353–2360.

Gur E, Sauer RT. (2008) Evolution of the ssra degradation tag in mycoplasma: specificity switch to a different protease. Proceedings of the National Academy of Sciences,105(42):16113– 16118.

Cameron DE, Collins JJ. (2014) Tunable protein degradation in bacteria. Nature Biotechnology, 32(12):1276–1281.

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.

William and Mary iGEM 2017 - https://parts.igem.org/Part:BBa_K2333011

Codon Optimisation Tool - www.jcat.de